ES2573229T3 - Cysteine protease inhibitors - Google Patents

Abstract

A compound of Formula II: ** Formula ** wherein R2 is the side chain of leucine, isoleucine, cyclohexylglycine, O-methyl threonine, 4-fluoroleucine or 3- methoxivaline; R3 is H, methyl or F; Rq is CF3 with the indicated stereochemistry and Rq 'is H; or Rq and Rq 'jointly define keto; Q is where R4 is C1-C6 alkyl; R5 is H, methyl or F; R6 is C1-C6 alkyl; or a pharmaceutically acceptable salt, a hydrate or an N-oxide thereof.

Description

DESCRIPTION

Cystem protease inhibitors Field of the invention

This invention relates to cystem protease inhibitors, especially those of the papafine superfamily. The invention provides novel compounds useful in the prophylaxis or treatment of disorders that result from an imbalance of physiological proteases, such as cathepsin K.

Description of the related technique

The superfamily of the cystem protease papama is widely distributed in various species that include mairnferos, invertebrates, protozoa, plants and bacteria. Numerous mai ^ ero cathepsin enzymes, which include cathepsins B, F, H, K, L, O and S, have been included in this superfamily, and inappropriate regulation of their activity has been implicated in a variety of metabolic disorders. including arthritis, muscular dystrophy, inflammation, glomerulonephritis and tumor invasion. Cathepsin-like pathogenic enzymes include bacterial gingipams, malaria falcipams I, II, III and following, and the cystem proteases of Pneumocystis carinii, Trypanosoma cruzei and brucei, Crithidia fusiculata, Schistosoma spp.

15 Inappropriate regulation of cathepsin K has been implicated in several disorders including osteoporosis, gingival diseases such as gingivitis and periodontitis, Paget's disease, hypercalcemia due to malignant disease and metabolic osteopathy. Due to its elevated levels in the chondroclasts of the osteoarthritis synovial membrane, cathepsin K is implicated in diseases characterized by excessive degradation of the cartilage or matrix, such as osteoarthritis and rheumatoid arthritis.

20 The treatment of bone and cartilage disorders, such as osteoarthritis and osteoporosis, may require a lifetime administration of a cathepsin K inhibitor, often to a population of patients in or near the geriatric phase. This implies unusually high requirements to facilitate the administration of the drugs intended for such disorders. For example, attempts are being made to extend the dosing regimes of the current osteoporosis drugs, of the bisphosphonate class, to regimens of 25 weekly or more extensive administrations to help continue the treatment. However, even with an improved dosage, other side effects of bisphosphonates still exist. Bisphosphonates block bone renewal instead of attenuating it as does a cathepsin K inhibitor. In order to have healthy bones, it is important to maintain the remodeling process that bisphosphonates completely block. In addition, bisphosphonates have a very long half-life in the bone, so that if effects such as osteonecrosis of the mandible are manifested, it is impossible to remove the bisphosphonate from the bone. In contrast, cathepsin K inhibitors normally have a rapid association and dissociation mode of action, which means that if a problem is to be identified, the dosage can be stopped and there is no accumulation of the inhibitor in the bone matrix.

Therefore, alternative drugs against osteoporosis and osteoarthritis with superior pharmacokinetic and / or pharmacodynamic properties are desired.

Compounds of formula IA are described in international patent application No. WO02 / 057270:

image 1

where UVWXY corresponds broadly to P3 and P2 (these expressions are explained below) of inhibitors of cystem protease dipeptides, Z is among others O, S, methylene or -NR-, R'i is alkyl, alkylaryl, etc. and Pi and Q are each, among others, methylene. Although the generic description in this patent application postulates a very wide range of substituents in Pi and Q, none is individualized or exemplified and no guidance on its synthesis is provided. In fact - the only synthetic suggestions provided in WO02 / 05720 do not allow a substitution in Pi or Q at all. The compounds are supposed to be useful, inter alia, for the treatment of protozoal infections such as trypanosomes.

Example 9 of International Patent Application Document No. WO2005 / 066180 discloses, among other things, a compound of formula IB:

5

10

fifteen

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The compound is an active inhibitor of cathepsin K, but, as shown below, the additional modification of the structure produces improvements in relation to pharmacokinetics and / or pharmacodynamics, in particular improved stability in whole blood and , therefore, a better exposure.

WO2008 / 007127, which had not been published on the priority date of the present application, describes compounds of formula IC:

image3

which are described as cystem protease inhibitors, in particular, cathepsin K inhibitors. Brief description of the invention

In accordance with the present invention, a compound of Formula II is provided:

image4

where

R2 is the side chain of leucine, isoleucine, cyclohexylglycine, O-methyl threonine, 4-fluoroleucine or 3- methoxivaline;

R3 is H, methyl or F;

Rq is CF3 with the indicated stereochemistry and Rq 'is H; or Rq and Rq 'jointly define keto;

What is it

image5

where

R4 is C1-C6 alkyl;

5

10

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25

R5 is H, methyl or F;

R6 is C1-C6 alkyl;

or a pharmaceutically acceptable salt, a hydrate or an N-oxide thereof.

According to one aspect of the invention, enantiomeric compounds of formula are provided:

image6

where

R2 is the side chain of leucine, isoleucine, cyclohexylglycine, O-methyl threonine, 4-fluoroleucine or 3- methoxivaline;

R3 is H, methyl or F;

R4 is C1-C6 alkyl, preferably methyl;

R5 is H, methyl or F;

or a pharmaceutically acceptable salt, an N-oxide or a hydrate thereof.

An alternative embodiment of the invention comprises pharmaceutically acceptable compounds or salts, N-oxides or hydrates of compounds of formula IIb

image7

wherein R2 and R3 are as defined above and Q is the N-alkyl-piperazinyl-thiazolyl moiety defined in formula IIa or the 4- (C1-C4 alkyl) sulfonylphenyl moiety, as described in WO 07/006716, for example a compound of the formula:

image8

The term "C1-C6 alkyl" means an alkyl chain having between one and six carbon atoms (for example, a C1-4 alkyl group, or a C1-3 alkyl group). The alkyl groups can be straight or branched chain. Suitable C1-6 alkyl groups include, for example, methyl, ethyl, propyl (for example, n-propyl and isopropyl), butyl (for example, n-butyl, iso-butyl, sec-butyl and tert-butyl), pentyl (for example, n-pentyl) and hexyl (for example, n-hexyl). An alkyl group of particular interest is methyl.

It will be appreciated that the compounds of the invention may exist as hydrates, such as those of the formulas for

Cial:

image9

and the invention extends to all these alternative forms.

In a preferred embodiment of the invention, R2 is the side chain of leucine, isoleucine, O-methyl threonine, fluoroleucine or 3-methoxivaline.

Currently preferred values of R2 include those illustrated by partial structures:

image10

especially the R2 value corresponding to the side chain of L-leucine.

The embodiments of the immediately preceding paragraph are advantageously applied to compounds in which R 3 or R 5 10 are fluoro.

fifteen

Normally the variable R3 such as methyl or fluoro, if present, is located in the target position with respect to the blessed amide bond, as shown below in the partial structure:

image11

A representative compound of this embodiment has the formula:

image12

In certain embodiments of the invention, R5 is F, especially when R3 is H and / or R4 is methyl. A representative compound of this embodiment has the formula:

image13

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Other preferred embodiments, with the stereochemistry depicted above include:

N- [1- (6-Chloro-3-oxo-hexahydro-furo [3,2-b] pyrrole-4-carbonyl) -3-fluoro-3-methyl-butyl] -4- [2- (4- methyl-piperazin-1-yl) -

thiazol-4-yl] -benzamide;

N- [1- (6-Chloro-3-oxo-hexahydro-furo [3,2-b] pyrrole-4-carbonyl) -3-fluoro-3-methyl-butyl] -4- [5-methyl-2 - (4-methyl-piperazin-

1-yl) -thiazol-4-yl] -benzamide;

N- [1- (6-Chloro-3-oxo-hexahydro-furo [3,2-b] pyrrole-4-carbonyl) -3-fluoro-3-methyl-butyl] -4- [5-fluoro-2 - (4-methyl-

piperazin-1-yl) -thiazol-4-yl] -benzamide;

N- [1- (6-Chloro-3-oxo-hexahydro-furo [3,2-b] pyrrole-4-carbonyl) -3-fluoro-3-methyl-butyl] -3-methyl-4- [2 - (4-methyl-piperazin-

1-yl) -thiazol-4-yl] -benzamide;

N- [1- (6-Chloro-3-oxo-hexahydro-furo [3,2-b] pyrrole-4-carbonyl) -3-fluoro-3-methyl-butyl] -3-fluoro-4- [2 - (4-methyl-

piperazin-1-yl) -thiazol-4-yl] -benzamide;

N- [1- (6-Chloro-3-oxo-hexahydro-furo [3,2-b] pyrrol-4-carbonyl) -3-fluoro-3-methyl-butyl] -3-methyl-4- [5 -methyl-2- (4-methyl-

piperazin-1-yl) -thiazol-4-yl] -benzamide;

N- [1- (6-Chloro-3-oxo-hexahydro-furo [3,2-b] pyrrol-4-carbonyl) -3-fluoro-3-methyl-butyl] -3-methyl-4- [5 -fluoro-2- (4-methyl-

piperazin-1-yl) -thiazol-4-yl] -benzamide

N- [1- (6-Chloro-3-oxo-hexahydro-furo [3,2-b] pyrrole-4-carbonyl) -3-fluoro-3-methyl-butyl] -3-fluoro-4- [5 -methyl-2- (4-methyl-

piperazin-1-yl) -thiazol-4-yl] -benzamide;

N- [1- (6-Chloro-3-oxo-hexahydro-furo [3,2-b] pyrrole-4-carbonyl) -3-fluoro-3-methyl-butyl] -3-fluoro-4- [5 -fluoro-2- (4-methyl-

piperazin-1-yl) -thiazol-4-yl] -benzamide;

and pharmaceutically acceptable salts, hydrates and N-oxides thereof.

Preferred embodiments of the invention include the compounds of formula II indicated:

N- [1-6-Chloro-3-oxo-hexahydro-furo [3,2-5] pyrrol-4-carbonyl) -3-fluoro-3-methyl-butyl] -4- [2- (4-methyl -piperazin-1-il) -

thiazol-4-yl] benzamide;

N- [2- (6-Chloro-3-oxo-hexahydro-furo [3,2-6] pyrrol-4-yl) -1-cyclohexyl-2-oxo-ethyl] -4- [2- (4- methyl-piperazin-1-yl) -thiazol-4-

il] -benzamide

N- [1- (6-Chloro-3-oxo-hexahydro-furo [3,2-6] pyrrole-4-carbonyl) -3-methyl-butyl] -4- [2- (4-methyl-piperazin- 1-yl) -thiazol-4-

il] benzamide

N- [1- (6-Chloro-3-oxo-hexahydro-furo [3,2-b] pyrrole-4-carbonyl) -3-methyl-butyl] -4- [5-methyl-2- (4- methyl-piperazin-1-yl) -thiazol-4-yl] -benzamide; or

a pharmaceutically acceptable salt, a hydrate or an N-oxide thereof.

Particularly preferred embodiments of the invention include the compound of formula II indicated N- [1- (6-chloro-3-oxo-hexahydro-furo [3,2-b] pyrrol-4-carbonyl) -3-methyl-butyl] -3-fluoro-4- [2- (4-methyl-piperazin-1-yl) -thiazol-4-yl] -benzamide; or a pharmaceutically acceptable salt, a hydrate or an N-oxide thereof.

Particularly preferred embodiments of the invention include the compound of formula II indicated N- [1- (6-chloro-3-oxo-hexahydro-furo [3,2-b] pyrrol-4-carbonyl) -3-methyl-butyl] -4- [5-fluoro-2- (4-methyl-piperazin-1-yl) -thiazol-4-yl] -benzamide; or a pharmaceutically acceptable salt, a hydrate or an N-oxide thereof.

Additional aspects of the invention include a pharmaceutical composition comprising a compound as defined above and a pharmaceutically acceptable carrier or diluent thereof.

A further aspect of the invention is the use of a compound as defined above for the treatment or preparation of a medicament for the treatment of disorders mediated by cathepsin K, such as:

osteoporosis,

gingival diseases such as gingivitis and periodontitis,

Paget's disease,

hypercalcemia due to malignant disease,

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metabolic bone disease,

diseases characterized by excessive degradation of the cartilage or matrix, such as osteoarthritis and rheumatoid arthritis,

bone cancers including neoplasia,

pain, especially chronic pain.

The compounds of the invention can form salts that form an additional aspect of the invention. Pharmaceutically acceptable salts of the compounds of Formula II include salts of organic acids, especially carboxylic acids, including but not limited to acetate, trifluoroacetate, lactate, gluconate, citrate, tartrate, maleate, malate, pantothenate, isethionate, adipate, alginate , aspartate, benzoate, butyrate, digluconate, cyclopentanate, glucoheptanate, glycerophosphate, oxalate, heptanoate, hexanoate, fumarate, nicotinate, palmoate, pectinate, 3-phenylpropionate, picrate, pivalate, propionate, tartrate, tartrate, canrtrate, tartrate, canrtrate, tartrate, cantarate, tartrate, cantarate, tartrate, cantarate, tartrate, cantarate, tartrate, cantarate, tartrate, cantarate, tartrate, cantarate, tartrate, canrtrate, cantarate, tartrate, cantarate, tartrate, cantarate, tartrate, cantarate, tartrate, cantarate, cantarate and succinate, organic sulfonic acids such as methanesulfonate, ethanesulfonate, 2-hydroxyethane sulfonate, camphorsulfonate, 2-naphthalenesulfonate, benzenesulfonate, p-chlorobenzenesulfonate and p-toluenesulfonate; and inorganic acids such as hydrochloride, hydrobromide, iodhydrate, sulfate, bisulfate, hemisulfate, thiocyanate, persulfate, phosphoric and sulfonic acids.

The compounds of the invention can be isolated in some cases in the form of hydrate. Hydrates are normally prepared by recrystallization from a mixture of aqueous / organic solvent using organic solvents such as dioxin, tetrahydrofuran or methanol. Hydrates can also be generated in situ by administration to a patient of the corresponding ketone.

The N-oxides of compounds of the invention can be prepared by methods known to those of ordinary skill in the art. For example, the N-oxides can be prepared by treating an oxidized form of the compound of the invention with an oxidizing agent (for example, trifluoroperacetic acid, permaleic acid, perbenzoic acid, peracetic acid, meta-chloroperoxybenzoic acid or the like) in a suitable inert organic solvent (for example, a halogenated hydrocarbon such as dichloromethane) at about 0 ° C. Alternatively, the N-oxides of the compounds of the invention can be prepared from the N-oxide of an appropriate starting material.

Examples of N-oxides of the invention include those with partial structures:

image14

The compounds of the invention in non-oxidized form can be prepared from N-oxides of the corresponding compounds of the invention, by treatment with a reducing agent (for example, sulfur, sulfur dioxide, triphenylphosphine, lithium borohydride, borohydride of sodium, phosphorus bichloride, tribromide or the like) in a suitable inert organic solvent (for example, acetonitrile, ethanol, aqueous dioxane or the like) from 0 to 80 ° C.

It should be noted that the positions of the radicals in any molecular moiety used in the definitions can be anywhere in that moiety, provided they are chemically stable.

The radicals used in the definitions of the variables include all possible isomers unless otherwise indicated.

When any variable appears more than once in any component, each definition is independent.

Unless otherwise mentioned or indicated, the chemical name of a compound includes the mixing of all possible stereochemically isomeric forms that said compound may possess. Said mixture may contain all diastereomers and / or enantiomers of the basic molecular structure of said compound. All stereochemically isomeric forms of the compounds of the present invention, both in pure form and mixed with each other, are understood to be included within the scope of the present invention.

The pure stereoisomeric forms of the compounds and intermediates mentioned herein are defined as isomers substantially free of other enantiomeric or diastereomeric forms of the same basic molecular structure of said compounds or intermediates. In particular, the term "stereoisomerically pure" refers to compounds or intermediates having an excess of stereoisomer of at least 80% (ie, a minimum of 90% of an isomer and a maximum of 10% of the others). isomers

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possible) up to a 100% stereoisomeric excess (that is, 100% of one isomer and none of the other), more particularly, compounds or intermediates that have a stereoisomeric excess of 90% to 100%, even more particularly that they have a stereoisomeric excess from 94% to 100% and most particularly that they have a stereoisomeric excess from 97% to 100%. The terms "enantiomerically pure" and "diastereomerically pure" should be similarly understood, but then taking into account the enantiomeric excess and the diastereomeric excess, respectively, of the mixture in question.

The compounds of the invention can be prepared as their individual stereoisomers by reacting a racemic mixture of the compound with an optically active resolving agent to form a pair of diastereoisomeric compounds, separating the diastereomers and recovering the optically pure enantiomer. Although the resolution of enantiomers can be carried out using covalent diastereoisomeric derivatives of the compounds of Formula (I), dissociable complexes (eg, crystalline; diastereoisomeric salts) are preferred. Diastereomers have different physical properties (for example, melting points, boiling points, solubilities, reactivity, etc.) and can be easily separated by taking advantage of these differences. The diastereomers can be separated by chromatography, for example HPLC or, preferably, by separation / resolution techniques based on differences in solubility. The optically pure enantiomer is then recovered, together with the resolving agent, through any suitable means that does not give rise to racemization. A more detailed description of the techniques applicable to the resolution of stereoisomers of compounds from their racemic mixture can be found in Jean Jacques Andre Collet, Samuel H. Wilen, Enantiomers, Racemates and Resolutions, John Wiley & Sons, Inc. (1981 ).

Although it is possible for the active agent to be administered alone, it is preferable to present it as part of a pharmaceutical formulation. Such formulation will comprise the active agent defined above together with one or more acceptable vegetables / excipients and optionally other therapeutic ingredients. The link (s) must be acceptable in the sense that they are compatible with the other ingredients of the formulation and not be harmful to the recipient.

The formulations include those suitable for rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration, but preferably the formulation is an orally administered formulation. The formulations can be conveniently presented in unit dosage form, for example, sustained release tablets and capsules, and can be prepared through any of the methods well known in the art of pharmacy.

Such methods include the step of associating the active agent defined above with the bond. In general, the formulations are prepared by uniformly and intimately associating the active agent with finely divided liquid or solid bonds, or both and then, if necessary, shaping the product. Methods for preparing a pharmaceutical composition comprising introducing a compound of Formula II or its pharmaceutically acceptable salt together or in association with a pharmaceutically acceptable carrier or carrier are disclosed. If the preparation of pharmaceutical formulations involves the thorough mixing of pharmaceutical excipients and the active ingredient in salt form, then it is normally preferred to use excipients that are not of a basic nature, that is, acidic or neutral.

Formulations for oral administration in the present invention may be presented as discrete units such as capsules, seals or tablets, each containing a predetermined amount of the active agent; as powder or granules; as a solution or suspension of the active agent in an aqueous liquid or a non-aqueous liquid; or as a liquid emulsion of oil in water or a liquid emulsion of water in oil, and as a bolus, etc.

With regard to compositions for oral administration (eg, tablets and capsules), the appropriate vein expression includes links such as common excipients, for example, binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (Povidone ), methyl cellulose, ethyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, sucrose and starch; fillers and bonds, for example, corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, caolm, mannitol, dicalcium phosphate, sodium chloride and alginic acid; and lubricants such as magnesium stearate, sodium stearate and other metal stearates, stearic acid, glycerol stearate, silicone fluid, waxes, talc, oils and colloidal silica. Flavoring agents such as mentha, gaulteria oil, cherry flavor or the like can also be used. It may be desirable to add a coloring agent to make the dosage form easily identifiable. The tablets can also be coated by methods well known in the art.

A tablet can be prepared by compression or molding, optionally with one or more accessory ingredients. The tablets may be prepared by compressing in a suitable machine the active agent in the form of free flow such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surfactant or dispersing agent. Molded tablets may be prepared by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or grooved and may be formulated to provide a slow or controlled release of the active agent.

Other formulations suitable for oral administration include lozenges comprising the agent

active in a flavored base, usually sucrose and gum arabic or tragacanth; tablets comprising the active agent in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active agent in a suitable liquid vehicle.

The appropriate dosage for the compounds or formulations of the invention will depend on the indication and on the patient and is easily determined by conventional tests on animals and is confirmed by clinical trials on humans. Dosages that provide intracellular concentrations (for the inhibition of physiological proteases of the papama superfamily) of the order 0.01 to 100 jM, more preferably 0.01-10 | jM, such as 0.1-25 | jM, They are normally desirable and feasible.

The compounds of the invention are prepared through a variety of chemical reactions in solution and in solid phase.

The compounds are usually prepared as basic elements that reflect residues P1, P2 and P3 of the final inhibitor product. Without wishing to be bound by the theone, or assign provisional union modes for specific variables, the theoretical concepts P1, P2 and P3 as used herein are provided for convenience only and have substantially the conventional meaning of Schlecter & Berger and indicate the portions of the inhibitor that are believed to fill subsites S1, S2 and S3, respectively, of the enzyme, where S1 is adjacent to the cleavage site and S3 is distanced from the cleavage site. The compounds defined by Formula II are understood to be within the scope of the invention, regardless of the mode of binding.

In general terms, the basic element P1 will have the formula:

image15

20 where

PG is a conventional N-protecting group or the free amine;

the two Rb groups define a ketal, such as bis methyl ketal or together define a cyclic ketal such as 1,3-dioxolane;

and Rc is a hydroxy protecting group, or less commonly is H or represents the keto function of the inhibitor 25 of the final product, in cases where the basic element P1 is the ketone, which is elongated with P2 and P3.

P2 is normally an L-leucine, L-isoleucine, O-methyl-L-threonine, L-3-hydroxivaline, 4-fluoroleucine or L-protected N-cyclohexylglycine, and P3 normally comprises a termination group, such as a derivative of benzoic acid with the N-alkyl-piperazinyl-E moiety already introduced or provided with a tune for it in the position for.

The individual basic elements, suitably protected, can be prepared first and then coupled to each other subsequently, preferably with the sequence P2 + P1 ^ P2-P1 followed by N-alkyl-piperazinyl-thiazolyl-benzoic acid * + P2-P1 ^ N-alkyl -piperazinyl-thiazolyl-benzoate-P2-P2-P1, where * indicates an activated form, in order to minimize racemization in P2.

The coupling between two amino acids, an amino acid and a peptide, or two peptide fragments can be carried out using conventional coupling procedures such as the azide method, the mixed carbonic-carboxylic acid anhydride method (chloroformate) of isobutyl), the carbodiimide method (dicyclohexylcarbodiimide, diisopropylcarbodiimide or carbodiimide water soluble), the active ester method (p-nitrophenyl ester, N-hydroxysucdonic ester imide), Woodward K reagent method, the method of carbonyldiimidazole, phosphorus reagents or oxidation-reduction methods. Some of these methods (especially the carbodiimide method) can be improved by adding 1-hydroxybenzotriazole or 4-DMAP. These coupling reactions can be carried out in solution (liquid phase) or in solid phase.

More explicitly, the coupling step involves the dehydration coupling of a free carboxyl of one reagent with the free amino group of the other reagent, in the presence of a coupling agent to form the bond with an amide bond. Descriptions of such coupling agents are found in general textbooks on peptide chemistry, for example, M. Bodanszky, "Peptide Chemistry", 2nd ed. rev., Springer-Verlag, Berlin, 45 Germany (1993), hereinafter referred to simply as Bodanszky, the contents of which are incorporated herein by reference. Examples of suitable coupling agents are N, N'-dicyclohexylcarbodiimide, 1- hydroxybenzotriazole in the presence of N, N'-dicyclohexylcarbodiimide or N-ethyl-N '- [(3-dimethylamino) propyl] carbodiimide. A

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Practical and useful coupling agent is commercially available (benzotriazol-1-yloxy) tris (dimethylamino) phosphonium hexafluorophosphate, either by itself or in the presence of 1-hydroxybenzotriazole or 4-DMAP. Another practical and useful commercially available coupling agent is 2- (IH-benzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium tetrafluoroborate. Yet another practical and useful coupling agent is O- (7-azabenzotriazol-1-yl) - N, N, N ', N'-tetramethyluronium hexafluorophosphate, commercially available.

The coupling reaction is carried out in an inert solvent, for example, dichloromethane, acetonitrile or dimethylformamide. An excess of a tertiary amine is added, for example, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine or 4-DMAP to maintain the reaction mixture at a pH of about 8. The reaction temperature is usually between 0 ° C and 50 ° C, and the reaction time is normally between 15 min and 24 h.

Functional groups of unnatural constituent amino acids should generally be protected during coupling reactions to avoid the formation of unwanted bonds. Protective groups that can be used are detailed in Greene, "Protective Groups in Organic Chemistry", John Wiley & Sons, New York (1981) and "The Peptides: Analysis, Synthesis, Biology", vol. 3, Academic Press, New York (1981), hereinafter referred to simply as Greene, whose descriptions are incorporated herein by reference.

The alpha-carboxyl group of the C-terminal residue is generally protected as an ester that can be cleaved to provide the carboxylic acid. Protective groups that may be used include: 1) alkyl esters such as methyl, trimethylsilyl and tert-butyl, 2) aralkyl esters such as benzyl and substituted benzyl, or 3) esters that can be cleaved with a mild base or media mild reducers such as trichlorethyl and phenacyl esters.

The alpha-amino group of each amino acid to be coupled is normally protected in N. Any protecting group known in the art can be used. Examples of such groups include: 1) acyl groups such as formyl, trifluoroacetyl, phthalyl and p-toluenesulfonyl; 2) aromatic carbamate groups such as benzyloxycarbonyl (Cbz or Z) and substituted benzyloxycarbonyls, and 9-fluorenylmethyloxycarbonyl (Fmoc); 3) aliphatic carbamate groups such as tert-butyloxycarbonyl (Boc), ethoxycarbonyl, diisopropylmethoxycarbonyl and allyloxycarbonyl; 4) dicalkyl carbamate groups such as cyclopentyloxycarbonyl and adamantyloxycarbonyl; 5) alkyl groups such as triphenylmethyl and benzyl; 6) trialkylsilyl such as trimethylsilyl; and 7) thiol-containing groups such as phenylthiocarbonyl and dithiasuccinyl. The preferred alpha-amino protecting group is either Boc or Fmoc. Many amino acid derivatives suitably protected for peptide synthesis are commercially available.

The alpha-amino protecting group is normally cleaved before the next coupling step. When the Boc group is used, the methods of choice are trifluoroacetic acid, pure or dichloromethane, or HCl in dioxane or ethyl acetate. The resulting ammonium salt is then neutralized either before coupling or in situ with basic solutions such as aqueous buffers, or tertiary amines in dichloromethane or acetonitrile or dimethylformamide. When the Fmoc group is used, the reagents of choice are piperidine or piperidine substituted in dimethylformamide, but any secondary amine can be used. The deprotection is carried out at a temperature between 0 ° C and the ambient temperature usually from 20 to 22 ° C.

Once the inhibitor sequence has been completed, any remaining protective group is removed in any manner dictated by the choice of protective groups. These procedures are well known to those skilled in the art.

The first stage in the synthesis of the compounds of the invention, such as those of the general formula II, is usually the preparation in solution of a functionalized basic element P1, for example, as shown in the following scheme:

image16

i) MsCI, Pyr ii) NaOAc, AC2O, DMF, 130 C iii) BFaxEt2O, EtaSiH, DCM iv) TBDPS-Cl, Im-H, DMF v) NaOMe vi) SO2CI2, Pyr, DCM vii) PPh3, MeOH, H2O , then EtaN, 50 C viii) B0C2O, EtaN ix) TBAF, THF x) Dess-Martin periodian xi) method a: MeOH, TMOF, p-TsOH, then EtaN, B0C2O, then chromatography and, 5 finally, MeOH, AcCI, method b: MeOH, AcCI, TMOF

Although the above scheme has been illustrated with a different protective group strategy using acetyl, mesyl, TBDPS and Boc, it will be apparent that other permutations of conventional protecting groups can be employed, as described by Greene (ibid). In addition, it may be convenient to use the ketone group dimethyl hemiacetal during the coupling of residues P2 and P3 and to regenerate the ketone function at a later stage.

10 The elongation of the basic element with the basic elements P2 and P3 is normally carried out in the presence of a suitable coupling agent, for example, benzotriazol-1-yloxytrisphyrrolidinophosphonium hexafluorophosphate (PyBOP), O-benzotriazol-1-yl hexafluorophosphate -N, N, N ', N'-tetramethyl-uronium (HBTU), O- hexafluorophosphate (7- azabenzotriazol-1-yl) -1,1,3,3-tetramethyl-uronium (HATU), 1 hydrochloride - (3-dimethylaminopropyl) -3-ethylcarbodiimide (EDC) or 1,3-dicyclohexylcarbodiimide (DCC), optionally in the presence of 1-hydroxybenzotriazole (HOBT) and a

Base such as N, N-diisopropylethylamine, triethylamine, N-methylmorpholine and the like. The reaction is usually carried out between 20 and 30 ° C, preferably at about 25 ° C, and requires 2 to 24 hours to complete. Suitable reaction solvents are inert organic solvents, such as organic halogenated solvents (for example, methylene chloride, chloroform and the like), acetonitrile, N, N-dimethylformamide, ethereal solvents such as tetrahydrofuran, dioxane and the like.

Alternatively, the coupling stage by elongation above can be carried out by first converting the basic element P3 / P2 into an active acid derivative such as succinimide ester and then doing it

react with the P1 amine. The reaction normally requires 2 to 3 hours to complete. The conditions used in this reaction depend on the nature of the active acid derivative. For example, if it is an acid chloride derivative, the reaction is carried out in the presence of a suitable base (for example triethylamine, diisopropylethylamine, pyridine and the like). Suitable reaction solvents are polar organic solvents such as acetonitrile, N, N-dimethylformamide, dichloromethane or any suitable mixture thereof.

The basic elements P2 in the form of N-protected L-amino acids are commercially available, for example, L-leucine, L-isoleucine, L-cyclohexylglycine, O-methyl-L-threonine and others are commercially available with numerous variants of protecting groups, such as CBz, Boc or Fmoc. Other variants of R2 are easily prepared from commercially available starting materials. For example compounds in which R2 is -

10 C (CHa) 2OCHa can be prepared by reacting the protected acid (S) - (+) - 2-amino-3-hydroxy-3-methylbutanoic acid

with CBz with 3,3-dimethoxy-hexahydro-furo (3,2b) pyrrole to form the desired P2-P1 unit. The alcohol of the P2 side chain can then be methylated using methyl iodide under conventional conditions of sodium hydride, imidazole, THF to obtain the desired P2 without substantial racemization of the alpha center. This remainder P2-P1 can then advance through the synthesis, as described herein, that is, by removing CBz and coupling.

15 A synthesis of basic elements of Fmoc and N-Boc-gammafluoroleucine are shown in Truong et al., Synlett 2005 No. 8 1278-1280.

The preparation of the basic elements P3 is described in WO05 / 66180 or they are easily prepared by analog methods. For example, the following scheme shows the preparation of a fluoro substituted thiazolyl:

image17

20 i. HOAC, Br2, TA, 2 h, 55% yield; ii. KF, 18-crown-6, CH3CN, 90 ° C, 16 h, 31% yield; iii. HOAC, Br2, 45 ° C, 4 h, 100% yield; iv. 4-methylpiperazin-1-carbothioamide, ethanol, 70 ° C, 2 h, 74% yield, v. LiOH, THF, H2O, TA, 16 h, 79% yield.

Synthesis of 4- [5-fluoro-2- (4-methyl-piperazin-1-yl) -thiazol-4-yl] -benzoic acid

The starting material, methyl 4-acetylbenzoate, is commercially available. The bromination in position a of the ketone is achieved with bromine in acetic acid to provide the desired 4- (2-bromo-acetyl) -benzoic acid methyl ester. Subsequent treatment of the 4- (2-bromo-acetyl) -benzoic acid methyl ester with potassium fluoride in the presence of 18-crown-6 at 90 ° C, provides the 4- (2-fluoro-acetyl) acidic methyl ester. ) -benzoic after a column chromatography. Repeated bromination in the ketone position a is achieved with bromine in acetic acid to provide the desired 4- (2-bromo-fluoro-acetyl) -benzoic acid methyl ester. The formation of thiazole is normally carried out by heating the 4- (2-bromo-2-fluoro-acetyl) -benzoic acid methyl ester with 4-methylpiperazin-1-carbothioamide at 70 ° C for 2 hours. Upon cooling, the 4- [5- fluoro-2- (4-methyl-piperazin-1-yl) -thiazol-4-yl] -benzoic acid methyl ester precipitates. The deprotection of the methyl ester is carried out using a solution of lithium hydroxide and the desired acid, the acid 4- [5-fluoro-2- (4-methyl-piperazin-1-yl) -thiazol-4- yl] -benzoic acid is generally obtained in good yield as the dihydrochloride salt after additional reactions with chlortndric acid.

WO05 / 066159 and WO05 / 065578 describe the preparation of compounds in which units P2 and P3 are linked to each other through a C (CF3) moiety, including those in which P3 is a biphenylsulfone. An example of the preparation of a basic element P2-P3 of this type, suitable for the preparation of compounds of formula II, wherein Rq is trifluoromethyl and Rq 'is H, is shown in scheme 3.

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1) isopropyl alcohol,

h2so4

2) pinacolborane, [1,1 '-bis (diphenylphosphino) chloride - ferrocen] palladium (ll)

[1,1'-bis (diphenylphosphino) chloride - ferrocen] palladium (ll)

Scheme 3

The protection of the acid function of the bromine derivative (3a), prepared according to the procedure described in Bioorg. Med. Chem Lett. 2006, 16, 1985, by reaction with, for example, isopropyl alcohol in the presence of an acid such as sulfuric acid, provides the corresponding ester. A desired thiazole derivative can then be coupled to the aromatic group of the ester provided using, for example, Stille or Suzuki coupling conditions. For example, the ester of bromine derivative 3a can react with a borane reagent such as pinacolborane in the presence of [1,1'-bis (diphenylphosphino) ferrocen] palladium II chloride to provide the corresponding dioxoborolane derivative. Subsequent substitution of the boronic group with a desired thiazole derivative (3b) in the presence of [1,1'-bis (diphenylphosphino) ferrocen] palladium II chloride then provides the biaryl derivative (3c). The thiazole derivatives (3b) can be prepared, for example, as described in J. Med. Chem. 2005, 48, 75207534. The removal of the acid protecting group, for example, by treatment with an acid such as hydrochloric acid in dioxane, it provides the basic element P2-P3 ready for coupling with a basic element P1, to provide the compounds of the invention. WO07 / 006716 shows the preparation and coupling of basic elements P3 and P3-P2 of this type.

The term "N-protecting group" or "N-protected" as used herein refers to those groups intended to protect the N-terminal end of an amino acid or a peptide, or to protect an amino group against reactions. undesirable during synthetic procedures. Commonly used N protecting groups are described in Greene, "Protective Groups in Organic Synthesis" (John Wiley & Sons, New York, 1981), which is incorporated herein by reference. N-protecting groups include acyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, a-chlorobutyryl, benzophlo, 4-chlorobenzoHo 4-bromobenzoflo, 4-nitrobenzoflo and the like; sulfonyl groups such as benzenesulfonyl, p-toluenesulfonyl and the like, carbamate forming groups such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2- nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2- nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1- (p-biphenylyl) -1-methylethoxycarbonyl, a, a-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-butoxycarbonyl, diisopropylmethyl isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4- nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl; alkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl and the like; and silyl groups such as trimethylsilyl and the like. Preferred N protecting groups include formyl, acetyl, benzoflo, pivaloyl, t-butylacetyl, phenylsulfonyl, benzyl (bz), t-butoxycarbonyl (BOC) and benzyloxycarbonyl (Cbz).

The hydroxyl and / or carboxyl protecting groups are also extensively reviewed in Greene ibid and include ethers such as methyl ethers, substituted methyl ethers, such as methoxymethyl, methylthiomethyl, benzyloxymethyl, t-butoxymethyl, 2-methoxyethoxymethyl and the like, silyl ethers such as trimethylsilyl ethers (TMS), t-butyldimethylsilyl (TBDMS) tribencylsilyl, triphenylsilyl, t-butyldiphenylsilyl, triisopropylsilyl and the like, substituted ethyl ethers, such as, for example, 1-ethoxymethyl, 1-methyl-1-methyl ethers -methoxyethyl, t-butyl, allyl, benzyl, p-methoxybenzyl, diphenylmethyl, triphenylmethyl and the like, aralkyl groups such as trityl and pixyl (derivatives of 9-hydroxy-9-phenylxanthene, especially chloride). Hydroxy ester protecting groups include esters such as formate, benzylformate, chloroacetate, methoxyacetate, phenoxyacetate, pivaloate, adamantoate, mesitoate, benzoate and the like. Carbonate hydroxyl protecting groups include methyl vinyl, allyl, cinnamonyl, benzyl and the like.

Detailed description of the achievements

Various embodiments of the invention will be described below, only by way of illustration referring to the following Examples.

Example 1

Preparation of the basic element P1:

5 Stage a)

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1 (7.0 g, 28.5 mmol) (3-azido-3-deoxy-1,2-O- (1-methyl ethylidene) -aD-alofuranose, prepared as described in Tronchet, Jean MJ; Gentile , Bernard; Ojha-Poncet, Joelle; Moret, Gilles; Schwarzenbach, Dominique; Barbalat-Rey, Frangoise; Tronchet, Jeannine Carbohydrate Research (1977), 59 (1), 87-93) dissolved in dry pyridine (50 mL) and the solution was cooled to 0 ° C. Mesyl chloride was added slowly to the solution and the solution was allowed to warm to room temperature. The reaction was stirred overnight and after 14 h MeOH (10 mL) was added followed by EtOAc (150 mL). The solution was washed three times with 2M H2SO4 (aq) (3 x 100 mL) and twice with satd NaHCO3. (ac) (2 x 100 mL) and then the organic phase was dried with Na2SO4, filtered and concentrated in vacuo. After placing the product overnight in a high vacuum pump to remove residual solvents, product 2 was obtained as a pale yellow oil with quantitative yield (11.5 g).

1 H NMR (CDCl 3, 400 MHz) 1.34 (s, 3 H), 1.51 (s, 3 H), 3.07 (s, 3 H), 3.16 (s, 3 H), 4.18 (d, 1 H, J = 3.1), 4.36 (dd, 1 H, J = 8.6, 3.2), 4.42 (dd, 1H, J = 12.0, 5.0), 4 , 67 (dd, 1H, J = 11.9, 2.3), 4.74 (d, 1H, J = 3.7), 5.11 (ddd, 1H, J = 8.6, 5.0 , 2.3), 5.89 (d, 1H, J = 3.6).

Stage b)

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Compound 2 (11.5 g, 28.5 mmol) was dissolved in DMF (50 mL). NaOAc (23.4 g, 285 mmol) and Ac2O (48.6 mL, 0.514 mol) were added to the solution, which was then heated at 125 ° C for 86 h. A part of the solvent was removed by rotary evaporation before the addition of 500 mL of EtOAc. The very dark solution was filtered through Celite. The organic phase was then washed with H2O (3 x 350 mL). The organic phase was dried with Na2SO4, filtered and the solvent removed by rotary evaporation. The crude products were purified by flash column chromatography (heptane: ethyl acetate 7: 3 -> 2: 1) to provide the desired compound 3 in a yield of 61% (5.70 g) and compound 4 in a yield of 22% (2.34 g).

Compound 3: 1 H NMR (CDCh, 400 MHz) 1.34 (s, 3H), 1.53 (s, 3H), 2.09 (s, 3H), 2.11 (s, 3H), 3.94 (d, 1H, J = 3.4),

4.19 (dd, 1 H, J = 12.2, 5.0), 4.32 (dd, 1 H, J = 8.0, 3.3), 4.37 (dd, 1H, J = 12.3, 3.5), 4.73 (d, 1H, J = 3.6), 5.32-5.37

30 (m, 1H), 5.94 (d, 1H, J = 3.8).

Compound 4: 1 H NMR (CDCh, 400 MHz) 1.34 (s, 3H), 1.51 (s, 3H), 2.10 (s, 3H), 3.10 (s, 3H), 4.11 (d, 1H, J = 3.6),

4.21 (dd, 1H, J = 12.8, 6.2), 4.32 (dd, 1H, J = 8.3, 3.2), 4.65 (dd, 1H, J = 12, 7, 2.2), 4.73 (d, 1H, J = 3.5), 5.09 (ddd,

1H, J = 8.3, 6.1.2.3), 5.89 (d, 1H, J = 3.5).

Stage c)

Triethylsilane (27.6 mL, 173 mmol) was added to compound 3 (5.70 g, 17.3 mmol) dissolved in dry DCM (40 mL). The round bottom flask was placed under an inert atmosphere (N2) in an ice bath, and allowed to cool, before the

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slow addition of BF3.Et2O (23.1 mL, 173 mmol). The reaction proceeded slowly and was stirred for 3 days. After this time, the starting material was still present. The slow addition of NaHCO3 (sat, ac) (70 mL) was followed by the addition of tablespoons of solid NaHCO3 until gas development ceased. The aqueous phase was extracted with DCM (150 mL) and washed with NaHCO3 (sat, aq) (70 mL) subsequently adding NH4Cl (sat, aq) (70 mL). The organic phase was dried with Na2SO4, filtered and concentrated in vacuo. The crude product was purified by flash column chromatography (heptane: ethyl acetate (2: 1) to provide a yield of 41% (1.95 g) of compound 5. 0.69 g of unchanged starting material was also isolated. .

1 H NMR (CDCl 3, 400 MHz) 2.08 (s, 3 H), 2.11 (s, 3 H), 3.72 (dd, 1 H, J = 10.0, 2.6), 3.95 (dd , 1H, J = 4.6, 2.1), 4.174.27 (m, 3H), 4.37 (dd, 1H, J = 12.1, 3.7), 4.52-4.57 ( m, 1H), 5.26-5.31 (m, 1H).

10 Stage d)

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Imidazole (1.46 g, 21.4 mmol) was added to a solution of compound 5 (1.95 g, 7.14 mmol) in DMF (50 mL). TBDPSCl was added after a couple of minutes and the reaction was stirred at room temperature for one night. Ethyl acetate (200 mL) was added to the reaction and the solution was washed with 10% dichloric acid (ac) (3 x 50 mL) and 15 NaHCO3 (sat, aq) (50 mL). The organic phase was dried with Na2SO4, filtered and concentrated in vacuo. The raw product is

purified by flash column chromatography (heptane: ethyl acetate (4: 1) to provide 6 with a yield of 92% (3.39 g).

1H NMR (CDCl3, 400 MHz) 1.09 (s, 9H), 2.04 (s, 3H), 2.05 (s, 3H), 3.71 (dd, 1H, J = 4.3, 2 , 1), 3.78 (dd, 1H, J = 9.5, 2.2), 3.99 (dd, 1H, J = 9.6, 4.6), 4.12 (dd, 1H, J = 12.2, 5.1), 4.28-4.33 (m, 2H), 4.36-4.40 (m, 1H), 5.17-5.22 (m, 20 1H) , 7.37-7.51 (m, 6H), 7.58-7.74 (m, 4H).

Stage e)

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NaOMe (10 mL, 0.5 M in MeOH) was added to a solution of 6 (3.39 g, 6.63 mmol) dissolved in MeOH (60 mL). The reaction was stirred at room temperature for 2 h before neutralizing the solution by adding Dowex 25 50 WX8 (H + form) until a neutral pH was reached. The beads were filtered off and the solvent removed

by rotary evaporation followed by high ford. Product 7 was obtained with a quantitative yield (2.66 g).

1H NMR (CDCl3, 400 MHz) 1.08 (s, 9H), 3.64 (dd, 1H, J = 11.5, 5.5), 3.71 (dd, 1H, J = 11.4, 3.9), 3.73-3.77 (m, 2H), 3.85-3.90 (m, 1H), 3.95 (dd, 1H, J = 9.6, 4.7), 4.15 (dd, 1H, J = 6.1, 4.2), 4.39-4.43 (m, 1H), 7.37-7.51 (m, 6H), 7.58-7 , 74 (m, 4H).

30 Stage f)

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Compound 7 (1.4 g, 3.27 mmol), dissolved in chloroform (10 mL) and pyridine (4.77 mL, 58.9 mmol), cooled in a dry ice bath, acetone. SO2O2 (1.56 mL, 19.6 mmol) was added and the bath was removed thereafter. The reaction mixture stirred overnight and became darker over time. After 16 h, the reaction mixture was diluted with DCM (15 mL) and washed with 10% duric acid (aq) (15 mL) and NaHCO3 (sat, aq) (15 mL). The organic phase was dried with Na2SO4, filtered and concentrated in vacuo. The brown oil was dissolved in MeOH (10 mL) and approx. 0.5 mL of Nal (0.8% in MeOH: H2O (1: 1)) was added to the solution that was stirred for 15 minutes. Then, the solvent was evaporated and the crude product was purified by instantaneous column chromatography (hep-

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Tano: ethyl acetate (4: 1) to provide a 68% yield of compound 8.

1 H NMR (CDCh, 400 MHz) 1.10 (s, 9H), 3.80-3.85 (m, 2H), 3.89 (dd, 1 H, J = 12.1, 5.8), 3.96 (dd, 1H, J = 9.7, 3.8), 4.00 (dd, 1H, J = 12.3, 2.6), 4.15 (ddd, 1H, J = 9, 7, 5.9, 2.6), 4.32- 4.36 (m, 2H), 7.35- 7.52 (m, 6H), 7.58- 7.75 (m, 4H)

Stage g)

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PPh3 (882 mg, 3.36 mmol) was added to a solution of compound 8 (1.04 g, 2.24 mmol) dissolved in MeOH (50 mL) and H2O (5 mL). The reaction was stirred at room temperature overnight. LC-MS showed that there was no starting material but very little cycled product. TEA (9.38 mL, 67.2 mmol) and H2O (5 mL) were added to the solution that was heated to 50 ° C. After 4 h, LC-MS did not show amines without cycling. The solvent was evaporated and the crude product was purified by flash chromatography (heptane: ethyl acetate (3: 2)) to provide product 9 with a yield of 54% (0.49 g). LRMS (M + H) 402.

1H NMR (CDCh, 400 MHz) 1.06 (s, 9H), 2.71 (dd, 1H, J = 11.1, 10.4), 3.18 (dd, 1H, J = 11.2, 7.0), 3.73 (d, 1H, J =

4.7), 3.78 (dd, 1H, J = 9.8, 3.5), 3.84 (dd, 1H, J = 9.8, 2.0), 3.95 (ddd, 1H , J = 10.2, 7.1.4.1), 4.16-4.19 (m, 1H), 4.66 (dd, 1H, J = 4.4, 4.4), 7, 35-7.46 (m, 6H), 7.61-7.67 (m, 4H).

Stage h)

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BOC anhydride (0.52 g, 2.40 mmol) was added to a solution of compound 9 (0.48 g, 1.20 mmol) dissolved in 50 mL of MeOH: TEA (9: 1). The reaction was stirred overnight and then the solvent was removed by concentration in vacuo. The crude product was purified by flash column chromatography (heptane: ethyl acetate (4: 1-> 2: 1)) to provide product 10 with a quantitative yield (0.60 g).

1H NMR (CDCh, 400 MHz) 1.07 (s, 9H), 1.24-1.46 (m, 9H) *, 3.05 (dd, 1H, J = 10.4, 10.4), 3.56 (d, 1H, J = 9.7), 3,703.89 (m, 1H) *, 3.90-4.15 (m, 2H) *, 4.24-4.89 (m, 3H ) *, 7.34-7.47 (m, 6H), 7.59-7.78 (m, 4H). * Indicates rotameros.

Stage i)

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Tetrabutylammonium fluoride (1.79 mL, 1.79 mmol) was added to a solution of compound 10 (0.60 g, 1.19 mmol) dissolved in THF (12 mL). The reaction was stirred at room temperature for 3 h before removing the solvent by concentration in vacuo. The crude product was purified by instantaneous column chromatography (hetero: ethyl acetate (1: 1 -> 0: 1) and a yield of 94% (0.29 g) of compound 11 was obtained.

1 H NMR (CDCh, 400 MHz) 1.45-1.52 (m, 9H) *, 3.16-3.32 (m, 1H) *, 3.83-4.22 (m, 5H) *, 4.41-4.54 (m, 1H) *, 4,664.71 (m, 1H) *. * Indicates rotameros.

J stage)

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Dess-Martin periodinan (0.60 g, 1.42 mmol) was added to a solution of compound 11 (0.34 g, 1.29 mmol) dissolved in dry DCM. The reaction was stirred under N2 atmosphere for 2 h, when it was estimated that the reaction was completed by tlc. The solution was washed 3 times (3 x 20 mL) with a 1: 1 mixture of 10% Na2S2O3 (aq) and 5 NaHCO3 (sat, aq). The organic phase was dried with Na2SO4, filtered and concentrated in vacuo. The crude product was purified by flash chromatography (heptane: ethyl acetate (3: 1) to provide a yield of 84% (284 mg) of compound 12.

1H NMR (CDCh, 400 MHz) 1.48 (s, 9H), 3.45 (dd, 1H, J = 11.3, 9.0), 4.01-4.17 (m, 2H), 4 , 19-4.41 (m, 3H), 4.684.87 (m, 1H).

10 Stage k)

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A pre-mixed solution of AcCl (42 ^ L, 0.601 mmol) and MeOH (5 mL) was added to a solution of compound 12 (263 mg, 1.01 mmol). After stirring for 2 h, additional AcCl (0.98 mL, 14 mmol) was added and again after stirring for 16 h, additional AcCl (9.8 mL, 140 mmol) was added. The reaction was completed shortly thereafter, concentrated in vacuo and subsequently any residual solvent was removed by high vacuum to provide a crude yield of 103% (253 mg) of compound 13.

1H NMR (CDCla, 400 MHz) 3.34 (s, 3H), 3.40 (s, 3H), 3.76 (d, 1H, J = 10.6), 3.72-3.90 (m , 1H), 4.15 (d, 1H, J = 10.4), 4.34 (d, 1H, J = 4.6), 4.50-4.60 (m, 1H), 4.69 -4.75 (m, 1H), 4.83 (s, 1H).

Example 2

20 P-2 coupling with L-Leu

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DIEA (304 ^ L, 1.84 mmol) and BOC-Leu (117 mg, 0.506 mmol) were added to crude 13 (112 mg, 0.460 mmol), dissolved in DMF (6 mL). The reaction flask was cooled in an ice bath for 10 minutes before the addition of HATU (193 mg, 0.506 mmol). The reaction was stirred for 3 hours at room temperature before concentrating at vacuum. The crude residue was dissolved in CHCl3 (15 mL) and washed with 10% dichloric acid (aq) (10 mL) and NaHCO3 (sat, aq) (10 mL). The organic phase was dried with Na2SO4, filtered and evaporated. The crude product was purified by flash chromatography (heptane: ethyl acetate (2: 1-> 1: 1) to provide product 14 with a yield of 62% (121 mg).

Example 3

30 Basic element P3

4- [5-methyl-2- (4-methyl-piperazin-1-yl) -thiazol-4-yl-benzoic acid

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Stage a) 4-Cyanopropiophenone

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As described for the preparation of 4-cyanoacetophenone (Synth. Commun 1994, 887-890), a mixture of 4- bromopropyrophenone (5.65 g, 26.4 mmol), Zn (CN) 2 (1.80 g , 15.3 mmol) and Pd (PPh3) 4 (2.95 g, 2.6 mmol) was heated to reflux at 80 ° C in deoxygenated DMF (35 mL, stored on 4 A molecular sieves, bubbled with Ar before of use) for 18 h. The mixture was partitioned between toluene (100 mL) and 2 N NH4OH (100 mL). The organic phase was extracted with 2N NH4OH (100 mL), washed with saturated aqueous NaCl (2 x 100 mL), dried and concentrated in vacuo. A reaction at 10 mmol scale was performed similarly and the raw products were combined. Instant chromatography (330 g of silica, 6/1 of petroleum ether - EtOAc) provided the desired compound as a white solid (5.17 g, 89%).

1H NMR (CDCla) 8 ppm: 1.22 (t, 3H, J = 7.2 Hz), 3.00 (c, 2H, J = 7.3 Hz), 7.75 (d, 2H, J = 8.8 Hz), 8.03 (d, 2H, J = 8.4 Hz)

13 C NMR (CDCla) 8 ppm: 7.8, 32.1, 116.1, 117.9, 128.3, 132.4, 139.7, 199.2 Stage b) 4-Propionylbenzoic acid

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4-Cyanopropiophenone (4.67 g, 29.3 mmol) was refluxed with 2 N NaOH (90 mL, 180 mmol) and dioxane (90 mL) at 95 ° C overnight. The mixture was diluted with water (150 mL), washed with ether (75 mL), acidified to pH 2 with concentrated HCl, and extracted with ether (3 x 75 mL). The organic phase was washed with saturated aqueous NaCl (3 x 75 mL), dried and concentrated to provide a yellow solid (5.12 g, 98%).

1H NMR (CDCls + CD3OD) 8 ppm: 1.18 (t, 3H, J = 7.2 Hz), 2.99, (c, 2H, J = 7.1 Hz), 7.95 (d, 2H , J = 8.4 Hz), 8.08 (d, 2H, J = 8.8 Hz)

13 C NMR (CDCls) 8 ppm: 7.9, 32.1, 127.7, 130.0, 134.0, 140.0, 168.0, 200.8 Stage c) Methyl 4-propionylbenzoate

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The above benzoic acid (890 mg, 5 mmol), NaHCO3 (1.26 g, 15 mmol) and iodomethane (935 pL, 15 mmol) in DMF (10 mL) were stirred at RT overnight. The mixture was diluted with saturated aqueous NaCl (50 mL) and extracted with ether (3 x 50 mL). The organic phase was washed with water (50 mL), dried and concentrated. Instant chromatography (90 g of silica, 2/1 of petroleum ether - EtOAc) gave a white solid (744 mg, 77%).

1 H NMR (CDCls) 8 ppm: 1.24 (t, 3H, J = 7 Hz), 3.03 (c, 2H, J = 7 Hz), 3.95 (s, 3H), 8.0 and 8 , 12 (ABc, 4H)

Stage d) 4- (2-Bromopropionyl) methyl benzoate

Jj — h — COOMe O '-'

Methyl 4-Propionylbenzoate (744 mg, 3.87 mmol), pyrrolidone tribromhydrate (1.98 g) and 2-pyrrolidinone (380 mg, 4.5 mmol) in THF (38 mL) were heated to 50 ° C under nitrogen atmosphere for 3 h. The mixture was cooled, filtered, concentrated in vacuo, and then redissolved in ether (50 mL). The ether solution was washed successively with water (20 mL), saturated aqueous Na2S2O5 (20 mL), saturated aqueous NaCl (20 mL) and water (20 mL), dried and concentrated in vacuo to provide a yellow oil (1,025 g) which was used directly in the Hantzsch coupling. This material contains 91% of the desired bromoketone, 5% of the starting ketone and 4% of 4-bromo-1-butanol, as determined by 1 H NMR.

1 H NMR (CDCls) 8 ppm: 1.92 (d, 3H, J = 7 Hz), 3.96 (s, 3H), 5.28 (c, 1H, J = 7 Hz), 8.07 and 8 , 14 (ABc, 4H)

Stage e) 4- [2- (4-tert-Butoxycarbonylpiperazin-1-yl) -5-methylthiazol-4-yl] benzoic acid methyl ester

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All of the above a-bromoketone and the 4-thionocarbonylpiperazin-1-carboxylic acid tert-butyl ester (J. Med. Chem., 1998, 5037-5054, 917 mg, 3.73 mmol) were refluxed in 36 mL THF at 70 ° C for 2 h, under N2 atmosphere. The precipitated material was filtered and the filtrate was concentrated in vacuo to provide a yellow solid. Instant column chromatography (silica, 5/1 petroleum ether - EtOAc) provided 624 mg of light yellow solids. Chromatography of the precipitate (silica, 2/1 petroleum ether - EtOAc) provided another 32 mg of compound. The total yield is 44%.

1 H NMR (CDCla) 5 ppm: 1.46 (s, 9H), 2.43 (s, 3H), 3.42, (m, 4H), 3.54 (m, 4H), 3.90 (s , 3H), 7.68 and 8.04 (ABc, 4H). Stage f) 4- [2- (4-Ferc-Butoxycarbonylpiperazin-1-yl) -5-methylthiazol-4-yl] benzoic acid

10

The above methyl ester (564 mg, 1.35 mmol) was heated with 1.35 mL of 2N NaOH, 5 mL of THF and 3.65 mL of water at 60 ° C for 4 h. The reaction mixture was evaporated, poured into 20 mL of saturated aqueous NaCl and 20 mL of CH2Cl2, and then acidified to pH 3 with 5% citric acid, in an ice bath. The layers were separated and the organic phase was further extracted with 2 x 10 mL of CH2Cl2. The organic phases were combined, washed with water (10 mL), dried and concentrated in vacuo to provide a light yellow solid (537 mg, 98%).

1H NMR (CDCla) 5 ppm: 1.48 (s, 9H), 2.47 (s, 3H), 3.47 (m, 4H), 3.57 (m, 4H), 7.74 and 8, 12 (ABc, 4H).

13C NMR (CDCls) 5 ppm: 12.6, 28.3, 42.8, 48.1, 80.3, 119.1, 127.8, 128.2, 130.1, 140.5, 145, 6, 154.6, 167.2, 171.4.

LCMS: (M + H) + 404, (M - H) - 402.

Step g) 4- [5-methyl-2- (4-methyl-piperazin-1-yl) -thiazol-4-yl] benzoic acid

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The 4- [4- (4-carboxyphenyl) -5-methylthiazol-2-yl] -piperazin-1-carboxylic acid tert-butyl ester (0.421 mmol) was dissolved in 4M HCl in 1,4-dioxane, and stirred at room temperature for 1 h. Then, the solvent was removed in vacuo, and the acid residue 4- (5-methyl-2-piperazin-1-yl-thiazol-4-yl) -benzoic acid was suspended in methanol (10 mL) and treated with AcOH / AcONa buffer (pH ~ 5.5, 5 mL) and formaldeddo (0.547 mmol). The reaction mixture was stirred at room temperature for 1 h, then treated with NaCNBH3 (0.547 mmol) and stirred at room temperature overnight. Then, the solvent was removed in vacuo, and the residue was purified by column chromatography to provide the phyto compound (0.403 mmol, 95%).

MS (ES) m / z 318 (100%, [M + H] +).

Example 4

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Acetyl chloride (0.4 mL) was added dropwise to a solution of compound 14 (0.121 g, 0.288 mmol) in methanol (4 mL) at 0 ° C. The reaction mixture was then stirred at room temperature overnight, and then concentrated in vacuo. The residue was redissolved twice in dry DMF (5 mL) and concentrated to dryness, then dissolved again in DMF (6 mL). HCl of 4- [5-methyl-2- (4-methyl-piperazin-1-yl) -thiazol-4-yl] - acid was added

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benzoic acid (112 mg, 0.316 mmol) and DIEA (190 jL, 1.15 mmol) to the solution before cooling to 0 ° C and HATU (120 mg, 0.316 mmol) was added. The reaction was stirred for 3 hours at room temperature before removing the solvent by rotary evaporation. The crude mixture was dissolved in CHCl3 (15 mL) and washed with 10% citric acid (aq) (10 mL) and NaHCO3 (sat, aq) (10 mL). The organic phase was dried with Na2SO4, filtered and evaporated. The crude product was purified by flash column chromatography (ethyl acetate: acetone 1: 1 + 0.2% TEA) to provide the product as a colorless oil / solid with a yield of 84% (150 mg). LRMS (M + H) 620.

NMR (CDCl3, 400 MHz) for the majority rotamer (13: 1 mixture of rotamers): 0.98 (d, 3H, J = 6.5), 1.04 (d, 3H, J = 6.4), 1.55-1.89 (m, 3H), 2.34 (s, 3H), 2.42 (s, 3H), 2.49-2.54 (m, 2H), 3.25 (s, 3H), 3.43 (s, 3H), 3.45-3.51 (m, 3H), 3.72 (d, 1H, J = 10.5), 3.91 (d, 1H, J = 10.5), 4.05-4.15 (m, 1H), 4.46-4.53 (m, 1H), 4.59 (dd, 1H, J = 5.2, 5.1), 4.74 (d, 1H, J = 5.6), 4.96-5.04 (m, 1H), 6.83 (d, 1H, J = 8.0), 7.68 (d, 2H , J = 8.3), 7.79 (d, 2H, J = 8.5).

Example 5

image39

Compound 15 (137 mg, 0.220 mmol) was dissolved in 20 mL of TFA: H2O (97.5: 2.5) and stirred for 4 hours. The solvent was removed in vacuo and the crude product was previously absorbed on silica to purify it by instantaneous column chromatography (EtOAc: acetone (1: 2) with 0.2% TEA) to provide the product (lyophilized from dioxane) in the form of an off-white solid with a yield of 71% (90 mg).

NMR (CDCl3, 400 MHz) for the majority rotamer (10: 1 mixture of rotamers): 0.98 (d, 3H, J = 6.5), 1.02 (d, 3H, J = 6.2), 1.58-1.86 (m, 3H), 2.34 (s, 3H), 2.42 (s, 3H), 2.50-2.54 (m, 2H), 3.44-3, 53 (m, 2H), 3.68 (dd, 1H, J = 10.4,

8.8), 4.11 (d, 1H, J = 17.2), 4.29 (d, 1H, J = 17.3), 4.36-4.44 (m, 1H), 4.59 ( dd, 1H, J = 10.5, 7.1), 4.81 (d, 1H, J =

5.8), 4.87-4.97 (m, 2H), 6. 83 (d, 1H, J = 7.9), 7.68 (d, 2H, J = 8.4), 7.79 ( d, 2H, J = 8.5).

Example 6

Nr (S) -1 - ((3aS.6R.6aS) -6-chloro-3-oxo-hexahydro-furor3,2-51pyrrol-4-carbonyl) -3-methyl-butyl1-4-r2- (4- methyl-piperazin-1-yl) -

thiazol-4-illbenzamide

Stage a)

image40

CL

TO

o o

12

■ cx

or

31

To a stirred solution of crude compound 12 (approximately 0.56 mmol) in trimethyl orthoformate (0.8 mL) and methanol (3 mL) p-toluenesulfonic acid monohydrate (p-TsOH, 0.007 g, 0.037 mmol) was added , then heated at 50 ° C overnight. The reaction mixture was then monitored by TLC (3: 2 hexane-ethyl acetate, staining with ninhydrin). The reaction mixture was heated to 60 ° C and a total of 21 mg of p-TsOH was added for 4 h, which provided the ketal not protected with Boc as the major product (indicated by TLC). The reaction mixture was then cooled to room temperature and triethylamine (0.32 mL, 2.24 mmol) was added followed by di-ferc-butyl dicarbonate (0.184 g, 0.84 mmol). The reaction mixture was maintained at room temperature for 2.5 h, then concentrated in vacuo and pre-absorbed on silica. Instant chromatography

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column in the residue column (gradient gradient elution, ethyl acetate in hexane, 20-25%), followed by concentration of the appropriate fractions in vat and removal of the residual solvent by further drying in a ford line during One night, I provide the product 31 in the form of an off-white solid (0.069 g, 0.022 mmol, 40% in 2 steps).

NMR data (400 MHz, 298 K, CDCla): 1H, 8 1.47 (s, 9 H), 3.16 (m, 1 H), 3.30 (s, 3 H), 3.37 ( s, 3 H), 3.72 (d, 1 H, J 9.3 Hz), 3.86 (m, 1 H), 3.98 (m, 1 H), 4.15 (m, 1 H ), 4.44 (d, 1 H, J 5.4 Hz), 4.61 (m, 1 H).

Stage b)

image41

To a stirred solution of compound 31 (0.074 g, 0.24 mmol) in methanol (2.7 mL) at 0 ° C, acetyl chloride (0.3 mL) was added dropwise over 1 minute. The reaction mixture was monitored by TLC (hexane-ethyl acetate 3: 2 and dichloromethane-methanol 95: 5, staining with ninhydrin) and after 4.5 h, the starting material was completely consumed. The reaction mixture was then concentrated in vacuo, then re-dissolved in dioxane with a few drops of H2O and lyophilized. The amorphous whitish solid obtained and W- (ferc-butoxycarbonyl) -L-leucine monohydrate (0.066 g, 0.26 mmol) was dissolved in DMF (3 mL) and concentrated in vacuo. Then, the residue was redissolved in DMF (3 mL) and W-ethyldiisopropylamine (0.13 mL, 0.72 mmol) was added. To this solution was added N-0- (7-azabenzotriazol-1-yl) -W, W, W, W-tetramethyluronium hexafluorophosphate (HATU, 0.12 g, 0.31 mmol) at 0 ° C. The reaction mixture was maintained at 0 ° C for 40 min, and an additional 75 minutes at room temperature. The reaction mixture was then diluted with ethyl acetate (25 mL), washed successively with aq. 10% (3 x 15 mL) and saturated sodium hydrogen carbonate ac. (3 x 15 mL) and then dried (sodium sulfate), filtered and concentrated in vacuo. Instantaneous column chromatography on silica gel of the residue using 2: 1 hexane-ethyl acetate as eluent, followed by vacuum concentration of the appropriate fractions, provided compound 32b as a colorless syrup (0.089 g, 0.21 mmol , 88%) which was used directly in the next stage.

Stage c

image42

To a solution of compound 32b (0.089 g, 0.21 mmol) in methanol (2.7 mL) at 0 ° C, acetyl chloride (0.3 mL) was added dropwise over 0.5 minutes. The reaction mixture was then stirred at RT for 5.5 h (monitored with TLC: dichloromethane-methanol 95: 5, tined using cerium ammonium sulfate sulfate in 10% aq sulfuric acid), and then concentrated to ford. The residue was redissolved in dioxane (5 mL) and a small amount of water was added, and then the solution was lyophilized. The colorless amorphous solid obtained and the HBr salt of 4- [2- (4-methyl-piperazin-1-yl) thiazol-4-yl] benzoic acid (0.089 g, 0.23 mmol) was dissolved in DMF (3 mL ) then N-ethyldiisopropylamine (0.15 mL, 0.85 mmol) was added and then the solution was cooled to 0 ° C and HATU (0.105 g, 0.275 mmol) was added. The reaction mixture was stirred at 0 ° C for 1 h and an additional 1 h at room temperature (monitored with TLC: dichloromethane-methanol 95: 5, visualized with UV light and staining using cerium ammonium sulfate sulphide in sulfuric acid ac 10%). The reaction mixture was then diluted with ethyl acetate (25 mL), washed with 3: 1 saturated sodium hydrogen carbonate / brine (3 x 20 mL), then dried (sodium sulfate), filtered and concentrated to ford. Instantaneous column chromatography of the residue using stepwise gradient elution (methanol in dichloromethane, 0-5%), followed by vacuum concentration of the appropriate fractions and lyophilization from dioxane (5 mL) and a few drops of water, provided product 32c as a colorless amorphous solid (0.121 g, 0.20 mmol, 94%).

Stage d)

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At 32c (0.114 g, 0.188 mmol) a solution of 97.5: 2.5 TFA: water (6 mL) was added at RT, the solution obtained was monitored by LC-MS and after stirring for 2 h at room temperature The reaction mixture was concentrated in vacuo. The residue was re-dissolved in ethyl acetate (25 mL), washed with saturated sodium hydrogen carbonate aq. (3 x 15 mL) and brine (1 x 15 mL), then dried (sodium sulfate), filtered and concentrated in vacuo. The residue obtained as an amorphous solid was dissolved in DMSO-acetonitrile-water-dioxane (approx. 12 ml) and purified by preparative HPLC-MS (Column: Sunfire 19 x 100 mm (C-is), eluent A: acetate 10 mM ammonium in water, eluent B: 10 mM ammonium acetate in 9: 1 acetonitrile-water, gradient: 30% B to 80% B in 8 minutes, flow: 20 mL / min). The appropriate fractions were concentrated in vacuo. The residue was redissolved in dioxane with a few drops of water, frozen and lyophilized, providing compound 32d as a whitish yellow amorphous solid (0.055 g, 0.10 mmol, 52%). An alfiquot part of the product obtained was dissolved in CDCl3 and analyzed by NMR, which indicated that the product came out in the keto form (undetectable hydrate form) in a 9: 1 mixture of rotamers. NMR data refers to the majority rotamer. When analyzed by HPLC-MS, the hydrate form is the majority form.

NMR data (500 MHz, 293 K, CDCls): 1H, 8 0.96 (d, 3 H, CH3-CH), 1.02 (d, 3 H, CH3-CH) 1.62-1.78 (m, 3 H, CH (CH3) 2 and CH2CH (CHs) 2), 2.37 (s, 3 H, CH3-N), 2.56 (m, 4 H, 2 x CH2-N), 3 , 59 (m, 4 H, 2 x CH2-N), 3.70 (m, 1 H, CHH-CHCl), 4.13 (d, 1 H, CHH-O), 4.31 (d, 1 H, CHH-O), 4.42 (m, 1 H, CHCl), 4.60 (m, 1 H, CHH-CHCl), 4.82 (m, 1 H, CHCl-CH), 4.90 -4.96 (m, 2 H, CH-C = O and CHNH), 6.85-6.89 (m, 2 H, NH and thiazol-H), 7.78 (d, 2 H, Ar- H), 7.89 (d, 2 H, Ar-H). LR-MS: Calculated for C27H35ClN5O4S: 560.2. Found: 560.3 [M + H], Calculated for C27H37ClN5OaS: 578.2. Found: 578.3.

Example 7

N- [2 - ((3aS.6R.6aS) -6-Chloro-3-oxo-hexahydro-furo [3,2-81pyrrol-4-yl) -1-S-cyclohexyl-2-oxo-ethyl1-4 - [2- (4-methyl-piperazin-1-

il) -thiazol-4-yl1-benzamide

image44

Step a) Tert-butyl acid ester [2 - ((3aS, 6R, 6aS) -6-chloro-3,3-dimethoxy-hexahydro-furo [3,2-6] pyrrole-4-yl) -1- S- cyclohexyl-2-oxo-ethyl1-carbamic (7a)

The HCl salt of (3aS, 6R, 6aS) -6-chloro-3,3-dimethoxy-hexahydro-furo [3,2-6] pyrrole (13) (0.23 mmol) and Boc-cyclohexyl-Gly-OH (64.4 mg, 0.25 mmol) was coevaporated from DMF, redissolved in 3 mL of DMF, and cooled in an ice bath. DIEA (160 pL, 0.92 mmol) was added, followed by HATU (108 mg, 0.28 mmol). After 20 minutes, the mixture was stirred at RT for 2 h 20 min, and concentrated in vacuo. The residue was dissolved in EtOAc (10 mL), washed successively with 10% Doric acid (5 mL), saturated NaHCO3 (5 mL) and saturated NaCl (2 x 5 mL). The organic phase was dried (Na2SO4) and concentrated. Instant column chromatography (silica, 2/1 pentane - EtOAc) provided a white solid (86.6 mg, 84% yield).

LCMS [M + 23] + = 469

Stage b) N- [2 - ((3aS, 6R, 6aS) -6-Chloro-3,3-dimethoxy-hexahydro-furo [3,2-6] pyrrol-4-yl) -1-S-cyclohexyl- 2-oxo-ethyl] -4- [2- (4- methyl-piperazin-1-yl) -thiazol-4-yl] -benzamide (7b)

image45

Acetyl chloride (0.25 mL) was added to an ice-cold solution of the tert-butyl ester of above carbamic acid (86.6 mg, 0.194 mmol) in methanol (2.20 mL). The mixture was stirred at RT for 3 h 45 min and evaporated. Lyophilization from dioxane-water provided the HCl salt of unprotected amine which was then coevaporated first from DMF with the acid HBr salt 4- [2- (4-methyl-piperazin-1-yl) thiazole -4-yl] benzoic acid (83 mg, 0.22 mmol) and then re-dissolved in 2.5 mL of DMF. The mixture was cooled in an ice bath, DIEA (140 ^ L, 0.80 mmol) was added, followed by HATU (83.8 mg, 0.22 mmol). After 15 minutes, the mixture was stirred at RT for 2.5 h. The mixture was concentrated, dissolved in EtOAc (20 mL), washed successively with saturated NaHCO3 (10 mL) and saturated NaCl (2 x 10 mL). The organic phase was dried (Na2SO4) and concentrated. Instant column chromatography 10 (silica, CH2O2-MeOH-Et3N)) gave white solids (121.2 mg, 99% yield).

LCMS [M + 1] + = 632

Stage c) N- [2 - ((3aS, 6R, 6aS) -6-Chloro-3-oxo-hexahydro-furo [3,2-b1pyrrol-4-yl) -1-S-ticlohexil-2-oxo- ethyl] -4- [2- (4-methyl-piperazin-1-yl) -thiazol-4-yl] -benzamide (7c)

image46

The above ether dimethoxy (115 mg, 0.182 mmol) was deprotected in a TFA-water solution (97.5: 2.5 v / v, 6.0 mL) by stirring at RT for 2 h 15 min. The mixture was concentrated, dissolved in EtOAc (25 mL), washed successively with saturated NaHCO3 (3 x 15 mL) and saturated NaCl (15 mL). The organic phase was dried (Na2SO4) and evaporated. The crude material was purified by HPLC-MS (Sunfire PrepC18 OBD 5 ^ m 19 x 100 mm column; 60 to 80% gradient of B in A, mobile phases A 10 nM NH4OAc in water and 10 nM NH4OAc in 90% MeCN) to provide the final compound as a white solid (63 mg, 59% yield).

LCMS ES + = 604 (hydrate) and ES + = 586 (ketone)

1H NMR (500 MHz, CDCls) 5 ppm 7.90 and 7.78 (ABc, 2H each, 6.89 (s, 1H), 6.83 (d, 1H, NH), 4.91 (m, 1H), 4.86 (m, 1H), 4.69 (m, 1H), 4.62 (dd, 1H), 4.40 (m, 1H), 4.32 and 4.14 (ABc, 1H each), 3.72 (dd, 1H), 3.60 (m, 4H), 2.58 (m, 4H), 2.38 (s, 3H), 2.10 - 1.04 (m, 11 H)

25 Example 8

Nr (S) -1 - ((3aS, 6R, 6aS) -6-chloro-3-oxo-hexahydro-furo [3,2-b1pyrrol-4-carbonyl) -3-fluoro-3-methyl-butyl1-4 -r2- (4-methyl-

piperazin-1-yl) -thiazol-4-yl] benzamide

image47

Stage a)

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Acetyl chloride (0.4 mL) was added dropwise to a solution of compound 11 (60 mg, 0.228 mmol) in methanol (4 mL) at 0 ° C. The reaction mixture was stirred at room temperature for 6 hours, concentrated, dissolved again in 1,4-dioxane and lyophilized overnight. The residue was dissolved in 5 mL of DMF. Fluoro-BOC-Leu-OH (Truong et al Synlett 2005 No. 8 1279-1280, 50 mg, 0.201 mmol) and DIEA (133 pL, 0.802 mmol) were added to the solution before cooling to 0 ° C and HATU (80 mg, 0.211 mmol) was added. The reaction was stirred for 3 hours at room temperature before concentrating the solvent in vacuo. The product was dissolved in EtOAc (20 mL) and washed with NaHCO3 (sat, aq) (10 mL). The organic phase was dried with Na2SO4, filtered and concentrated in vacuo. The product was purified by flash chromatography (ethyl acetate) to provide product 42 with a yield of 99% (79 mg).

Stage b)

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Acetyl chloride (0.4 mL) was added dropwise to a solution of compound 42 (79 mg, 0.199 mmol) in methanol (4 mL) at 0 ° C. The reaction mixture was stirred at room temperature for 6 hours, concentrated, dissolved again in 1,4-dioxane and lyophilized overnight. The residue was dissolved in 5 mL of DMF. 4- [2- (4-methyl-piperazin-1-yl) -thiazol-4-yl] -benzoic acid HBr (76 mg, 0.198 mmol). DIEA (119 pL, 0.721 mmol) was added to the solution before cooling to 0 ° C and HATU (72 mg, 0.189 mmol) was added. The reaction was stirred for 3 hours at room temperature before removing the solvent in vacuo. The product was dissolved in CHCh (15 mL) and washed with NaH-CO3 (sat, aq) (10 mL). The organic phase was dried with Na2SO4, filtered and evaporated. The product was purified by instant chromatography (chloroform: ethanol 7: 3 + 0.1% TEA) to produce suitably pure 43 which

could use directly in the next stage.

Stage c)

image50

Compound 43 (104 mg, 0.198 mmol) (not pure) was dissolved in 9 mL of DCM: DMSO (2: 1). TEA (111 pL, 0.777 mmol) was added followed by SO3 * pyridine (48 mg, 0.299 mmol). The reaction was stirred at room temperature and was

trolo by LC-MS. After 4 hours, another portion (48 mg) of SO3 * pyridine was added and after 4 more hours, another portion. After 22 hours (overnight) an additional portion was added and after 2 more hours a final portion was added. The solution was poured into a separatory funnel with 40 mL of DCM and washed with 20 mL of NaHCO3 (sat, aq). The organic phase was dried with Na2SO4, filtered and concentrated in vacuo. The product was purified by semi-preparative HPLC on a Sunfire C18 column with mobile phases A (90:10 of H2O: acetonitrile, 10 mM NH4Ac) and B (10:90 of H2O: acetonitrile, 10 mM NH4Ac) starting at 40-75 % of B. The product was obtained as an off-white solid with a yield of 29% (30 mg).

NMR (CDCl3, 400 MHz): 1.37-1.52 (m, 6H), 2.10-2.30 (m, 2H), 2.37 (s, 3H), 2.58-2.63 (m, 2H), 3.57-3.62 (m, 2H), 3.73 (dd, 1 H, J = 10.5, 8.7), 4.10 (d, 1 H, J = 16.9), 4.28 (d, 1 H, J = 16.9), 4.43-4.5 (m, 1H), 4.71 (dd, 1 H, J = 35 10.6, 6.9), 4.78 (d, 1H, J = 6.0), 4.87-4.93 (m, 1H), 4.95-5.03 (m, 1H), 6.86 ( s, 1H), 7.37 (d, 1H, J = 7.3), 7.73 (d, 2H,

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J = 8.3), 7.82 (d, 2H, J = 8.6). LRMS (M + H) 578. Example 9

An alternative P3 basic element

HCl salt of 3-fluoro-4- [2- (4-methylpiperazin-1-yl) -thiazol-4-yl-benzoic acid Step a) methyl 4-Bromo-3-fluorobenzoate (9a)

image51

4-Bromo-3-fluorobenzoic acid (2.46 g, 11.2 mmol) was dissolved in MeOH (9 mL) and toluene (4 mL) and cooled in an ice bath. (Trimethylsilyl) diazomethane (11 mL, 2.0 M in hexanes, 22 mmol) was added dropwise until the yellow color persisted. The solution was stirred at room temperature for 40 minutes and then concentrated in vacuo. A second batch of carboxylic acid (2.43 g) was treated similarly. The crude product from the two batches was combined and subjected to instant chromatography (silica, 5/1 of pentane-EtOAc) to provide the methylene ester as white solids (4.92 g, 95% yield).

1H NMR (400 MHz, CDCla) delta ppm 7.77 (m, 1H), 7.71 (m, 1H), 7.64 (m, 1H), 3.93 (s, 3H).

Stage b) methyl 4-Acetoxy-3-fluorobenzoate (9b)

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F

Allyl chloride (105 pL, 1.28 mmol) and TFA (20 pL, 0.26 mmol) were added to a suspension of zinc powder (480 mg, 7.34 mmol) and anhydrous cobalt (II) bromide ( 96.6 mg, 0.44 mmol) in MeCN (4 mL), in an inert gas atmosphere. After stirring at room temperature for 10 min, the aryl bromide (1.003 g, 4.30 mmol dissolved in 5 mL of MeCN) from (a) was added, followed by acetic anhydride (0.45 mL, 4.79 mmol) and more MeCN (1 mL). The mixture was stirred overnight, quenched with 1M HCl (20 mL), and then extracted with EtOAc (3 x 20 mL). The organic phase was washed successively with saturated aqueous NaHCO3 (20 mL) and saturated NaCl (2 x 20 mL), dried (Na2SO4) and concentrated. Instant chromatography (silica, 6/1 to 4/1 of petroleum ether - EtOAc) provided recovered bromide (161.1 mg, 16%) and the desired ketone (white solids, 305.5 mg, 36%) .

NMR (CDCla) delta ppm: 1H (400 MHz) 7.94-7.86 (m, 2H), 7.80 (dd, 1H, J = 11.2, 1.6 Hz), 3.95 (s , 3H), 2.67 (d, 3H, J = 4.4 Hz); 19F (376 MHz) -109.2 (m); 13C (100 MHz) 195.4 (d, J = 3.7 Hz), 165.1 (d, J = 2.2 Hz), 161.6 (d, J = 255 Hz), 135.8 (d , J = 8.1 Hz), 130.7 (d, J = 2.9 Hz), 129.0 (d, J = 14 Hz), 125.2 (d, J = 3.6 Hz), 117 , 9 (d, J = 26 Hz), 52.7 (s), 31.4 (d, J = 7.3 Hz).

Stage c) methyl 4- (2-Bromoacetoxy) -3-fluorobenzoate (9c)

image53

F

THF (10 mL) and 2-pyrrolidinone (91 pL, 1.20 mmol) were added to a mixture of the ketone from b) (198 mg, 1.01 mmol) and pyrrolidone tribromhydrate (532 mg, 1.07 mmol). After heating at 60-65 ° C for 2 h, the mixture was concentrated in vacuo and then partitioned between EtOAc (20 mL) and saturated Na2S2O3 (10 mL). The aqueous phase was extracted with EtOAc (10 mL). The organic phases were combined, washed with saturated NaCl (2 x 10 mL), dried (Na2SO4) and concentrated. Instant chromatography (silica, 7/1 of petroleum ether - EtOAc) provided white solids (0.2634 g) that account for 84% of the desired bromide (as determined by 19F NMR peak integration).

NMR (CDCl3) 6 ppm: 1H (400 MHz) 7.93 (m, 1H), 7.88 (m, 1H), 7.79 (dd, 1H, J = 11.2, 1.6 Hz), 4.50 (d, 2H, J = 2.4 Hz), 3.94 (s, 3H); 19F (376 MHz) -108.4 (m).

Stage d) Methyl 3-Fluoro-4- [2- (4-methylpiperazin-1-yl) -thiazol-4-yl] benzoate (9d)

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EtOH (5.0 mL) was added to the above bromo ketone (193 mg, 0.70 mmol) and 4-methyl-piperazin-1- carbothioic acid amide (113 mg, 0.71 mmol) and the mixture was heated to 70 ° C for 2h 15 min. The precipitates were filtered, washed with EtOH fno, and dried in vacuo and characterized. The procedure was repeated on a larger scale for 1.75 g of bromo ketone (6.36 mmol).

NMR (1/1 CDCla-CD3OD) 8 ppm: 1H (400 MHz) 8.20 (m, 1H), 7.86 (dd, 1H, J = 8.4, 1.6 Hz), 7.76 ( dd, 1H, J = 11.4, 1.8 Hz), 7.38 (d, 1H, J = 2.4 Hz), 4.23 (a, 2H), 3.95, (s, 3H) , 3.65 (a, 4H), 3.32 (a, 2H), 2.98 (s, 3H); 19F (376 MHz) - 114.0 (m). LCMS [M + H] + = 336.

The precipitates of both preparations were combined and suspended in saturated NaHCO3 (50 mL). The mixture was extracted with EtOAc. The organic phase was washed with water, dried (Na2SO4), and evaporated to provide the title compound as cream-colored solids (1.76 g).

Step e) HCl salt of 3-fluoro-4- [2- (4-methylpiperazin-1-yl) -thiazol-4-yl] benzoic acid (9e)

image55

The methyl ester (1.76 g, 5.25 mmol) (9d) was heated at 80 ° C with 6 M HCl (40 mL) for 5.5 h. More 6M HCl (10 mL) was added and the mixture was heated at 90 ° C for 1 h 15 min. After cooling, the mixture was evaporated in vacuo and lyophilized from water to provide the final product as cream-colored solids with quantitative yield.

NMR (DMSO-d6) 8 ppm: 1H (400 MHz) 11.60 (a, 1H), 8.18 (t, 1H, J = 8.0 Hz), 7.82 (dd, 1H, J = 8 , 4, 1.6 Hz), 7.72 (dd, 1H, J = 12.0, 1.6 Hz), 7.48 (d, 1H, J = 2.8 Hz), 4.11 (m , 2H), 3.58 (m, 2H), 3.49 (m, 2H), 3.19 (m, 2H), 2.80 (d, 3H, J = 4.4 Hz); 19F (376 MHz) -113.5 (m); 13C (100 MHz) 168.9, 166.0, 159.0 (d, J = 250 Hz), 143.4, 131.4 (d, J = 8 Hz), 129.8, 125.8 (d , J = 11 Hz), 125.6, 116.6 (d, J = 24 Hz), 111.1 (J = 15 Hz), 51.1, 45.0, 41.9. LCMS [M + H] + = 322.

Example 10

N - [(S) -1 - ((3aS.6R.6aS) -6-Chloro-3-oxo-hexahydro-furo [3,2-b1-pyrrol-4-carbonyl) -3-methyl-butyl-1-3-fluoro -4- [2- (4-methyl-

piperazin-1-yl) -thiazol-4-yl1-benzamide

image56

Stage a) N - [(S) -1 - ((3S, 3aS, 6R, 6aS) -6-Chloro-3-hydroxy-hexahydro-furo [3,2-b] pyrrole-4-carbonyl) -3- methyl-butyl] -3-fluoro-4- [2- (4-methyl-piperazin-1-yl) -thiazol-4-yl] -benzamide

image57

This compound was prepared from tert-butyl ester of N - [(S) -1 - ((3S, 3aS, 6R, 6aS) -6-chloro-3-hydroxy-hexahydro-furo [3,2-b ] pyrrole-4-carboxylic acid by a series of deprotection steps (acetyl chloride, MeOH) and HATU-mediated coupling with Bocleucina initially and, finally, 3-fluoro-4- [2- (4- methyl) HCl salt -piperazin-1-yl) -thiazol-4-yl] benzoic prepared as described in Example 9.

LCMS: [M + H] + = 580; [M-H] - = 578. 19F NMR (376 MHz, CDCla + CD3OD) delta ppm -113.3 (m).

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Stage b)

Triethylamine (20 jL) and sulfur trioxide-pyridine complex (20 mg, 0.125 mmol) were added to a solution of the alcohol (16.6 mg, 0.03 mmol) of (a) in 0.9 mL of CH2Cl2 and 0 , 45 mL of DMSO and stirred at RT. After 2 h, more triethylamine (10 jL) and sulfur trioxide-pyridine complex (10 mg) were added and the mixture was stirred overnight to complete oxidation to the ketone. The mixture was diluted with CH2Cl2 and washed with saturated NaHCO3 followed by saturated aqueous NaCl. The organic phase was dried (Na2SO4) and evaporated to provide an oil. The crude material was purified by HPLC-MS (Sunfire PrepC-is jm 19 x 100 mm column; OBD 5 gradient from 30 to 80% B in A, mobile phases A 10 mM NH4OAc in water and 10 mM NH4OAc in 90% MeCN) to provide the title compound as white solids (4.4 mg).

NMR (CDCh) delta ppm: 1H (500 MHz, 2 rotamers observed, majority rotamer described) 8.22 (m, 1H, phenyl H5), 7.56-7.54 (m, 2H, phenyl H2 and H6), 7.21 (m, 1H, thiazole), 6.81 (d, 1H, J = 8.5 Hz, NH), 4.94-4.85 (m, 2H, NHCHC = O and ClCCHO), 4, 84 (d, 1H, J = 6.0 Hz, (O = C) NCHC = O)), 4.56 (dd, 1H, J = 10.5, 7.0 Hz), 4.42 (m, 1H, ClCH), 4.32 and 4.14 (ABq, 1H each), 3.70 (dd, 1H, J = 10, 9 Hz), 3.59 (m, 4H), 2.57 (m , 4H), 2.37 (s, 3H, NMe), 1.8-1.6 (m, 3H, CH2CHMe2), 1.03 (d, 3H, J = 6.0 Hz, i-Pr), 0.96 (d, 3H, J = 6.5 Hz, i-Pr); 19F (376 MHz) -112.9 (m, majority rotamer, 84%) and -113.2 (m, minority, 16%).

LCMS: 577.4 Da monoisotopic molar mass; ES + = 578.4 (M + H) +, 596.5 [M + H2O + H] +.

Example 11

An alternative P3 / P2 basic element

image58

Stage a) Isopropyl acid ester (S) -2 - [(S) -1- (4-bromo-phenyl) -2,2,2-trifluoro-ethylamino] -4-methyl-pentanoic acid (11 a)

r

To a stirred solution of (S) -2 - [(S) -1- (4-bromo-phenyl) -2,2,2-trifluoro-ethylamino] -4-methyl-pentanoic acid, prepared as shown Li, CS et al Bioorg. Med. Chem. Lett. 2006, 16, 1985, (1.80 g, 4.9 mmol) in isopropyl alcohol (100 mL), concentrated sulfuric acid (2 mL) was added. The resulting solution was heated at 80 ° C for 4 hours. The reaction mixture was allowed to cool and then concentrated under vacuum. The resulting oil was dispersed in CH2Cl2 (100 mL), washed with saturated NaHCO3 (2 x 50 mL), dried (MgSO4) and concentrated to give the title compound as a brown oil (1.77 g, 88%) MS [M + H] 412.

Step b) Isopropyl ester of acid (S) -4-methyl-2 - {(S) -2,2,2-trifluoro-1- [4- (4,4,5,5-tetramethyl- [1,3 , 2] dioxaborolan-2-yl) -phenyl] -ethylamino} -pentanoic acid (11b)

image59

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To a stirred solution of the bromoderivative 1 k (2.2 g, 5.36 mmol) in DMF (30 mL) was added bis (pinacolato) boron (2.0 g, 8.04 mmol), potassium acetate (1, 6 g 16.1 mmol) and [1,1'-bis (diphenylphosphino) ferrocen] palladium (II) chloride in 1: 1 complex with CH2Cl2 (0.438 g, 0.54 mmol). The resulting solution was sealed in a tube and heated in a microwave at 160 ° C for 20 minutes. The reaction mixture was allowed to cool to room temperature and then filtered through a short column of silica eluted with ethyl acetate (500 mL). The resulting solution was concentrated to

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Ford and the crude product was purified by C18 column reverse phase chromatography (H2O: MeCN, 50-100% gradient) to provide the title compound as a brown oil (0.920 g, 38%). MS [M + H] 458.

Step c) Isopropyl ester of acid (S) -4-meffl-2 - ((S) -2,2,2-trifluoro-1- {4- [2- (4-methyl-piperazin-1-yl) - thiazol-4-yl] -phenyl} - ethylamino) -pentanoic acid (11c)

image61

To a stirred solution of the 1 k borolane derivative (0.72 g, 1.57 mmol) in DMF: H2O (1: 1, 20 mL) 1- (4- bromo-thiazol-2-yl) -4 was added -methyl-piperazine (0.5 g, 1.89 mmol), sodium carbonate (0.2 g, 1.89 mmol) and [1,1'-bis (diphenylphosphino) ferrocen] palladium (II) chloride in 1: 1 complex with CH2Cl2 (0.129 g, 0.16 mmol). The resulting solution was sealed in a tube and heated in a microwave at 160 ° C for 20 minutes. The reaction mixture was allowed to cool and then diluted with CH2O2 (100 mL). The organic phase was separated, dried (MgSO4) and concentrated in vacuo. The crude product was purified by instantaneous column chromatography (ethyl acetate: MeOH, 9: 1) to provide the title compound as a dark red solid (0.150 g, 13%). MS [M + H] 513. Retention time 4.0 minutes 50-97 (NH3) 2CO310 mM: MeCN 6 min Gradient C12 Reverse phase.

Step d) Acid (S) -4-methyl-2 - ((S) -2,2,2-trifluoro-1- {4- [2- (4-methyl-piperazin-1-yl) -thiazol-4 -yl] -phenyl} -ethylamino) -pentanoic; hydrogen chloride (11d)

image62

To a stirred mixture of 2M hydrochloric acid and dioxane (1: 1, 10 mL) isopropyl acid ester (S) -4-methyl-2 - ((S) -2,2,2-trifluoro-1- was added {4- [2- (4-methyl-piperazin-1-yl) -thiazol-4-yl] -phenyl} -ethylamino) -pentanoic acid (0.15 g, 0.29 mmol), prepared as described by Palmer et al. in J. Med. Chem. 2005, 48, 7520-7534. The solution was heated for 20 hours at 100 ° C and then concentrated in vacuo to provide the film compound as a dark brown solid (0.14 g, 98%) that can be coupled to the basic element P1 and the ketone regenerated by any of the methods described above, usually without further purification. MS M-H 469.

Example 11A

An alternative P3 basic element

image63

i. AcOH, bromine, TA, 2 h, 55% yield; ii. KF, acetonitrile, 18-crown-6, 90 ° C, 16 h; 31% yield; iii. AcOH, bromine, 45 ° C, 4 h, 100% yield; iv. 4-Methyl-piperazin-1-carbothioic acid amide, A, 2 h, 74% yield; LiOH, TA, 16 h, 100% yield.

Availability of starting materials -

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Methyl 4-acetylbenzoate is available from Aldrich; 4-methyl-piperazin-1-carbothioic acid amide - 11 suppliers were found in SciFinder (eg, Chem Pur Products Ltd in Germany).

Stage a) 4- (2-Bromo-acetyl) -benzoic acid methyl ester

or

OMe O

Bromine (8.4 mmol) was added to a solution of 4-acetyl-benzoic acid methyl ester (8.4 mmol) in acetic acid (20 mL). The reaction was stirred at RT for 2 h, during which time the red color disappeared and a whitish precipitate formed. The product was collected by filtration and washed with methanol / fresh water (200 mL 1: 1) to provide a white powder (55%). 1H NMR (400MHz, CDCls) 3.98 (3H, s), 4.20 (2H, s), 8.02 (2H, d, J = 8Hz), 8.18 (2H, d, J = 8Hz) .

Stage b) 4- (2-Fluoro-acetyl) -benzoic acid methyl ester

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To a suspension of potassium fluoride (3.11 mmol) in acetonitrile (1 mL) 18-crown-6 (0.1 mmol) was added and the reaction was heated at 90 ° C for 30 minutes. 4- (2-Bromo-acetyl) -benzoic acid (1.56 mmol) was added and the reaction was heated at 90 ° C for 16 h. The reaction was diluted with water (10 mL) and extracted with ethyl acetate (3 x 20 mL). The product was purified on silica eluting with 5-15% ethyl acetate in isohexane to give a vach concentration of the desired fractions, the title product as a white solid (31%). 1H NMR (400MHz, CDCla) 3.98 (3H, s), 5.55 (2H, d, J = 50Hz), 7.95 (2H, d, J = 8Hz), 8.18 (2H, d, J = 8Hz).

Stage c) 4- (2-Bromo-2-fluoro-acetyl) -benzoic acid methyl ester

image66

To a suspension of 4- (2-fluoro-acetyl) -benzoic acid (1.19 mmol) in acetic acid (5 mL), bromine (1.19 mmol) was added. The reaction was heated at 45 ° C for 4 h, during which time a green solution formed. The reaction was concentrated in vach and azeotroped twice with toluene to produce the title compound as a green solid (100%). The product was used raw in the next stage. 1H rMn (400MHz, CDCh) 3.98 (3H, s), 7.04 (1H, s), 8.05-8.10 (4H, m).

Step d) 4- [5-Fluoro-2- (4-methyl-piperazin-1-yl) -thiazol-4-yl] -benzoic acid methyl ester

image67

4- (2-Bromo-2-fluoro-acetyl) -benzoic acid methyl ester (1.18 mmol) and 4-methyl-piperazin-1- carbothioic acid amide (1.18 mmol) were dissolved in ethanol (10 mL) The reaction was heated at reflux for 2 h. The reaction was cooled to RT causing the product to precipitate. The product was collected by filtration and washed with cold ethanol. The product was treated with aqueous sodium bicarbonate to provide the title compound as a colorless oil (74%). MS (ES +) 337 (M + H, 100%).

Step f) 4- [5-Fluoro-2- (4-methyl-piperazin-1-yl) -thiazol-4-yl1-benzoic acid dihydrochloride

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To a solution of 4- [5-fluoro-2- (4-methyl-piperazin-1-yl) -thiazol-4-yl] -benzoic acid (0.43 mmol) in tetrahydrofuran / water (2, 5 mL, 4: 1) lithium hydroxide (0.5 mmol) was added. The reaction was stirred at RT for 16 h. The reaction was concentrated in vacuo and clorlddric acid (2 N, 3 mL) was added causing the product to precipitate as a white solid. The product was collected by filtration to provide the title product as a white solid (79%). MS (ES +) 322 (M + H, 100%).

Example 12

N- [1- (6-Chloro-3-oxo-hexahydro-furo [3.2-b1-pyrrol-4-carbonyl) -3-methyl-butyl1-4- [5-fluoro-2- (4-methyl-piperazin-1 -il) -thiazol-4-

ill-benzamide

image69

Stage a) 6-benzyloxy-3-oxo-hexahydro-furo [3,2-b] pyrrole-4-carboxylic acid (12a)

image70

Dess-Martin periodinano (12.5 g, 30 mmol) was dissolved in DCM (250 mL). Compound 10 from WO07 / 066180 (7.4 g, 20 mmol) in DCM (50 mL) was added to a stirred solution of oxidant at RT under nitrogen atmosphere in 45 min. Once the reaction was considered to have been completed according to TLC, 10% aqueous Na2S2O3 (200 mL) was added and the mixture was stirred at RT for another 15 minutes. The two-phase system was transferred to a separatory funnel and extracted twice with EtOAc (200 mL and 100 mL, respectively). The combined organic phases were washed once with saturated aqueous NaHCO3 (100 mL) and brine (100 mL), dried over Na2SO4, filtered and the solvent was evaporated in vacuo, yielding the crude product 2 as a clear oil (7, 69 g); ESI +, m / z: 368 (M + +1).

Stage b) 6-benzyloxy-3,3-dimethoxy-hexahydro-furo [3,2-b] pyrrole-4-carboxylic acid (12b)

image71

Compound 12a (7.6 g) was dissolved in dry methanol (100 mL). Trimethyl orthoformate (30 mL) and pTsOH (0.2 g) were added at RT under a nitrogen atmosphere. The mixture was heated at 60 ° C for 8 hours. Once the reaction was considered complete according to TLC, it was cooled to RT and concentrated to steam. The crude product was purified by column chromatography on silica gel eluting with ethyl acetate-heptane (1: 4) to provide after concentration in vacuo, ketal 12b as a clear oil (5.9 g, 71% in 2 stages); eS | +, m / z: 382 (M + -OMe).

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Step c) Ferc-butyl acid ester (3aS, 6R, 6aS) -6-hydroxy-3,3-dimethoxy-hexahydro-furo [3,2-b] pyrrol-4-carboxylic acid (12c)

image72

A solution of compound 12b (2.5 g, 6.4 mmol) in methanol (60 mL) and Pd (OH) 2 (0.7 g) was stirred at RT under H2 atmosphere for 48 hours. The mixture was filtered and concentrated under vacuum. The residue (2.8 g, 14.8 mmol) was dissolved in 75 mL of a dioxane / water mixture (2: 1). A solution of 10% Na2CO3 (25 mL) was added dropwise at pH 9-9.5. The mixture was cooled to 0 ° C in an ice-water bath and Boc added in one portion. The reaction was stirred at RT overnight and the pH of the mixture was maintained at 9-9.5 by the addition of more 10% Na2CO3 solution, if necessary. The mixture was filtered and the solvent was removed under vacuum. The aqueous mixture was extracted with 3 x 100 mL of EtOAc, the combined organic phases were washed with 100 mL of water and 100 mL of brine, dried over Na2SO4, filtered and the solvent was evaporated to vach to provide 3.79 g of carbamate as a clear oil (89%), ESI +, m / z: 312 (M ++ Na).

Step d) Bendic acid ester (3aS, 6R, 6aS) -6-hydroxy-3,3-dimethoxy-hexahydro-furo [3,2-6] pyrrole-4-carboxylic acid (12d)

image73

To a stirred solution of compound 12c (3.8 g, 13.13 mmol) in CH2Cl2 (100 mL) 2M HCl in MeOH (50 mL) was added. The resulting solution was stirred overnight and then concentrated under vacuum and azeotroped with toluene (3 x 100 mL). The crude residue was dissolved in CH2Cl2 (100 mL), cooled to 0 ° C and pyridine (1.071 µL, 13.13 mmol) was added followed by dropwise addition of CbzCl (1.875 jL, 13.13 mmol) . The reaction was stirred at room temperature for 2 hours, then washed with 2M HCl (2 x 50 mL), saturated NaHCO3 (2 x 50 mL), dried (MgSO4) and concentrated. The residue was purified by flash column chromatography (5-100% isohexane: EtoAc) to obtain the title compound as a clear oil (2510 mg, 59%). MS M + H 324.

Step e) Bendic acid ester (3aS, 6R, 6aS) -6-methanesulfonyloxy-3,3-dimethoxy-hexahydro-furo [3,2-b] pyrrol-4-carboxylic acid (12e)

image74

To a stirred solution of compound 12d (500 mg, 1.55 mmol) in CH2Cl2 (20 mL) was added triethyl amine (332 jL, 2.32 mmol) and mesyl chloride (266 mg, 2.32 mmol). After stirring for 30 minutes, the reaction was washed with saturated NaHCO3 (1 x 20 mL), 2M HCl (1 x 20 m), dried with MgSO4 and concentrated to provide the title compound (655 mg, 99% ) as a yellow oil. MS M + H 402.

Step f) Bendic acid ester (3aS, 6S, 6aS) -6-chloro-3,3-dimethoxy-hexahydro-furo [3,2-6] pyrrole-4-carboxylic acid (12f)

image75

To a stirred solution of compound 12e (550 mg, 1.37 mmol) in DMF (30 mL) was added lithium chloride (721 mg, 13.7 mmol). After stirring for 120 minutes at 120 ° C, the reaction was concentrated under vacuum. The residue was diluted with CH2Cl2 (50 mL), washed with water (1 x 20 mL), dried with MgSO4 and concentrated under vacuum. The residue was purified by flash column chromatography (5-66% isohexane: EtOAc) to provide the compound of the

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Four. Five

title (330 mg, 72%) as a yellow oil. MS M + H 342, 344.

Step g) Ferc-bufflic acid ester [2- (6-chloro-3,3-dimethoxy-hexahydrofuro [3,2-b] pyrrol-4-carbonyl) -3-methylbutyl] -carbamic acid (12g)

6-Chloro-3,3-dimethoxy-hexahydro-furo [3,2-b1-pyrrol-4-carboxylic acid (68 mg, 0.20 mmol) acid ester was deprotected by catalytic hydrogenation using palladium on carbon at 10% and hydrogen at atmospheric pressure. After stirring for 2 hours, the suspension was filtered through Celite and the filtrate was evaporated on a rotary evaporator to provide crude 6-chloro-3,3-dimethoxy-hexahydrofuro [3,2-b] pyrrole, which it was coupled with N-Boleucine using HATU in the same manner as in the method described in Example 2, which provided the title compound (78 mg, 93%). MS m / z 421.2 (M + H) +.

Step h) N- [2- (6-Chloro-3,3-dimethoxy-hexahydrofuro [3,2-b] pyrrol-4-carbonyl) -3-methylbutyl] -4- [5-fluoro-2- (4 -methyl-piperazin- 1-yl) -thiazol-4-yl] -benzamide (12h)

The tert-bufflic acid ester [2- (6-chloro-3,3-dimethoxy-hexahydrofuro [3,2-b] pyrrol-4-carbonyl) -3-methylbutyl-carbamic acid (78 mg, 0.185 mmol) was deprotected under acidic conditions (acetyl chloride in methanol) as described in Example 4, the crude pyrrole hydrochloride intermediate was then coupled with the acid HCl salt 4- [5- fluoro-2- (4-methyl -piperazin-1-yl) -thiazol-4-yl] -benzoic using the HATU conditions as described in Example 2, which provided the title compound (98 mg, 85%). MS m / z 624.2 (M + H) +.

1H-NMR (400MHz, CDCla): 7.91 (d, 2H), 7.81 (d, 2H), 6.85 (d, 1H), 5.00 (m, 1H), 4.75 (d , 1H), 4.60 (m, 1H), 4.50 (dt, 1H), 4.12 (d, 1H), 3.92 (d, 1H), 3.75 (m, 1H), 3 , 49 (m, 4H), 3.48 (m, 1H), 3.45 (s, 3H), 3.28 (s, 3H), 2.62 (m, 4H), 2.42 (s, 3H), 1.85 (m, 1H), 1.70 (m, 2H), 1.05 (d, 3H), 1.00 (d, 3H).

Stage i) N- [1- (6-Chloro-3-oxo-hexahydro-furo [3,2-b] pyrrol-4-carbonyl) -3-methylbutyl] -4- [5-fluoro-2- (4 -methyl-piperazin-1-yl) - thiazol-4-yl] -benzamide (12i)

N- [2- (6-Chloro-3,3-dimethoxy-hexahydrofuro [3,2-b] pyrrole-4-carbonyl) -3-methylbutyl] -4- [5-fluoro-2- (4-ethylpiperazin- 1-yl) -thiazol-4-yl] -benzamide (98 mg, 0.157 mmol) was hydrolyzed under acidic conditions as described in Example 5. The residue was purified by preparative HPLC chromatography (C8, gradient 10 - 90% MeCN / H2O) which gave the pure title compound 41.8 mg (46%), as a mixture of 2 rotamers, of both ketone (27%) [MS m / z 578.1 (M + H) +] and hydrate (73%) [MS m / z 596.1 (M + H2O + H) +].

Example 13

6-Chloro-4- (4-methyl-2- [2.2.2-trifluoro-1- (4'-methanesulfonyl-biphenyl-4-yl) -ethylamino1-pentanoyl} -tetrahydro-furo [3,2-b1-pyrrole- 3-

ona

image76

Step 1- (6-Chloro-3,3-dimethoxy-hexahydro-furo [3,2-b] pyrrol-4-yl) -4-methyl-2- [2,2,2-trifluoro-1- ( 4'-methanesulfonyl-biphenyl-4- yl) -ethylamino] -pentan-1-one (13a)

The 6-chloro-3,3-dimethoxy-hexahydro-furo [3,2-b] pyrrol-4-carboxylic acid (55 mg, 0.16 mmol) acid ester was deprotected by catalytic hydrogenation using palladium on carbon at 10% and hydrogen at atmospheric pressure. After stirring for 2 hours, the suspension was filtered through Celite and the filtrate was concentrated. The obtained amine was then coupled with 4-methyl-2- [2,2,2-trifluoro-1- (4'-methanesulfonyl-biphenyl-4-yl) -ethylamino] -pentanoic acid (76 mg, 0.17 mmol ), prepared as described in WO07 / 006716, using the HATU conditions as described in Example 2, which provided the title compound (101 mg, 50%).

Stage b

Compound 13a (47 mg, 0.07 mmol) was hydrolyzed under acidic conditions as described in Example 5. The residue obtained was purified by column chromatography (EtOAc-P. Ether 3: 2), which provided the pure title compound (26 mg), [MS m / z 586.4.

Example 14

6-Chloro-4- [4-methyl-2- (2.2.2-trifluoro-1- (4- [2- (4-methyl-piperazin-1-yl) -thiazol-4-yl-phenyl-ethylamino) -pentanoyl1-tetrahydro-

furo [3.2-b1pyrrol-3-one

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Step a) 1- (6-Chloro-3,3-dimethoxy-hexahydro-furo [3,2-b] pyrrol-4-yl) -4-methyl-2- (2,2,2-trifluoro-1- {4- [2- (4-methyl-piperazin-1- yl) -thiazol-4-yl] -phenyl} -ethylamino) -pentan-1-one (14a)

The benzyl group of the 6-chloro-3,3-dimethoxy-hexahydro-furo [3,2-b] pyrrole-4-carboxylic acid (35 mg, 0.10 mmol) benzyl ester was removed by catalytic hydrogenation using palladium on 10% carbon and hydrogen at atmospheric pressure. After stirring for 2 hours, the suspension was filtered through Celite and the filtrate was concentrated. The obtained amine was then coupled with the acid of Example 11, step D (42 mg, 0.09 mmol) using the HATU conditions as described in Example 2, which provided the title compound (45 mg).

Stage b

Compound 14a (47 mg, 0.07 mmol) was hydrolyzed under acidic conditions, as described in Example 5. The residue obtained was purified by column chromatography (CH2Cl2-acetone 2: 1 + 0.05% DIEA) what the pure title compound provided (20 mg).

Example 15

Alternative synthesis of the basic element P1

image78

Stage i)

Step g) of Example 1 was optimized as follows: A mixture of the monodyl and bidcyclic amines depicted above (approximately 1.8 mmol) of the first reaction of step g), was dissolved in ethyl acetate (25 mL) and triethylamine (1.5 mL) was added. The solution was heated at reflux for 3 h, monitored by LC-MS, then additional triethylamine (1.5 mL) was added and the reaction mixture was refluxed another 15 h. The reaction mixture was then cooled to about 0 ° C and benzyl chloroformate (0.38 mL, 2.7 mmol) was added in a portion and then allowed to reach RT. The reaction was monitored by TLC (4: 1 and 3: 2 hexane-ethyl acetate, visualized with UV light and staining with AMC) and after 4 h the reaction mixture was diluted with ethyl acetate (15 mL), it was washed successively with acf. 10% (3 x 25 mL) and saturated sodium hydrogen carbonate ac. (3 x 25 mL), then dried (sodium sulfate), filtered and concentrated. Instantaneous chromatography of the residue (gradient gradient elution, ethyl acetate in hexane, 10-20%), followed by a concentration of the appropriate fractions and drying in a vacuum overnight, provided the product as a colorless foam (0.57 g, 1.06 mmol).

NMR data (400 MHz, 298 K, CDCl3): 1H, 8 1.02 and 1.10 (2 s, 9H, C (CH3) 3), 3.13 (m, 1H, CHH), 3.59 and 3.80 (2 m, 2 x 1H, CH2), 3.99-4,1.5 (m, 2H, CHH and CH), 4.34, 4.42, 4.46 and 4.68 ( 4 sa, 2H, majority and minor CH), 4.84 (m, 1H, CH), 4.92-5.16 (m, 2H, CH2), 7.11-7.80 (m, 15 H, ArH).

Stage ii)

image79

To a stirred solution of the product from step i) (0.56 g, 1.05 mmol) in THF (6 mL) a solution of 1 M tetrabutylammonium fluoride in THF (1.26 mL) was added, and stirred to TA for one night. The reaction mixture was then concentrated on silica and an instantaneous column chromatography of the residue (stepwise gradient elution, ethyl acetate in hexane, 50-100%), followed by a concentration of the appropriate fractions and drying. overnight, I provide the product as a colorless syrup (0.27 g, 0.91 mmol, 87%).

NMR data (400 MHz, 298 K, CDCla): 1H, 8 2.22 and 3.00 (2 d, 1H, Joh3 = 3.5 Hz, majority and minor OH), 3.30 (m, 1 H , CHH), 3.89 (m, 1 H, CHH), 4.00-4.16 (m, 3H, 2 CHH and CH), 4.24 (d, 1H, CH), 4.43 and 4 , 54 (2 s, 1 H, majority and minor H-3), 4.70 (m, 1 H, CH), 5.08-5.23 (m, 2H, OC ^ Ph), 7.32-7, 40 (m, 5H, Ar-H).

Stage iii)

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To a stirred solution of the product from step ii) (0.26 g, 0.88 mmol) in dichloromethane (6 mL), Dess-Martin periodine (0.41 g, 0.97 mmol) was added at RT . The reaction was monitored by TLC (3: 2 ethyl acetate-hexane, visualized by staining with AMC) and after 3.5 h the reaction mixture was diluted with dichloromethane (20 mL), washed with 1: 1 hydrogen sodium carbonate ac. saturated / 10% sodium thiosulfate ac. (3 x 20 mL), then dried (sodium sulfate), filtered and concentrated. The residue was redissolved in methanol (5 mL) and trimethyl orthoformate (1.25 mL) and p-toluenesulfonic acid monohydrate (0.03 g, 0.16 mmol) were added. The reaction mixture was kept at 60 ° C overnight, then diisopropylethylamine (0.5 mL) was added and the reaction mixture was concentrated. Instant chromatography (gradient gradient elution, ethyl acetate in hexane, 20-40%) of the residue, followed by concentration of the appropriate fractions and drying in a vacuum during the weekend, provided the product as a syrup colorless hard (0.27 g, 0.79 mmol, 89%).

NMR data (400 MHz, 298 K, CDCl3): 1H, 8 3.08-3.47 (m, 7H, 2 x majority and minor OCH3 and CHH), 3.80 (m, 2H, CH2), 3 , 98 (sa, 1H, CH), 4.25 (m, 1H, CHH), 4.45 (m, 1H, CH), 4.60 (t, 1H, CH), 5.04-5.26 (m, 2H, CH2), 7.29-7.42 (m, 5H, Ar-H)

Comparative Example 1

25 N - [(S) -1 - ((3aS, 6S, 6aS) -6-chloro-3-oxo) -hexahydro-furo [3,2-6] pyrrole-4-carbonyl) -3-methyl-butyl ] -4- [2- (4-methyl-piperazin-1-yl) -

thiazol-4-yl] benzamide

image80

image81

image82

i. Dess-Martin newspaperman, DCM, 2 h, TA;

ii. Trimethylortoformate, pTsOH, MeOH, 8 h, 60 ° C;

iii. Pd (OH) 2, H2, MeOH, 48 h, RT;

5 iv. Boc2O, 10% Na2CO3, 16 h, 0 ° C to RT;

v. HCl, CH2CH2 / Py, CBzCl

saw. CH2CH2 / Et3N MsCl

vii. DMF, LiCI

This basic element was deprotected in N a continuation and carried through the rest of the synthesis as described in Example 5 to provide N - [(S) -1 - ((3aS, 6aS) -6R-chlorine -3-oxo) -hexahydro-furo [3,2-jb] pyrrole-4-carbonyl) -3-methyl-butyl] -4- [2- (4-methyl-piperazin-1-yl) -thiazol-4 -il] benzamide.

Comparative Example 2

N - [(1S) -1 - ((3aS, 6aR) -3-oxo) -hexahydro-furo [3,2-b] pyrrol-4-carbonyl) -3-methyl-butyl] -4- [2- (4-methyl-piperazin-1-yl) -thiazol-4-

il] benzamide

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image83

The basic element P1 was synthesized as described in WO02 / 05720, coupled to N-protected L-leucine and the basic element P3 of Example 3 above, and oxidized to give the ketone as shown in Example 4 .

Comparative Example 3

N - [(1S) -1 - ((3aS, 6aR) -3-oxo) -hexahydro-furo [3,2-b] pyrrol-4-carbonyl) -3-methyl-butyl] -4- [2- (4-methyl-piperazin-1-yl) -thiazol-4-

il] benzamide

image84

The synthesis of this comparative example is shown in Example 9 of WO05 / 66180.

Biological examples

Determination of the proteolytic catalytic activity of cathepsin K

Suitable assays for cathepsin K were carried out using a human recombinant enzyme, such as that described in PDB.

ID BC016058 standard; MRNA; HUM; 1699 BP.

DE cathepsin K of Homo sapiens (pycnodysostosis), mRNA (cDNA clone MGC: 23107 RX MEDLINE; RX PUBMED; 12477932.

DR RZPD; IRALp962G1234.

DR SWISS-PROT; P43235

Recombinant cathepsin K can be expressed in a variety of commercially available expression systems, including the E. coli, Pichia and Baculovirus systems. The purified enzyme is activated by the elimination of prosequence by conventional methods.

The standard test conditions for the determination of the kinetic constants used a fluorogenic peptide substrate, typically HD-Ala-Leu-Lys-AMC, and were determined in either 100 mM Month / Tris, pH 7.0 1 mM EDTA and 10 mM 2-mercaptoethanol or 100 mM Na phosphate, in 1 mM EDTA, 0.1% PEG4000 pH 6.5 or 100 mM Na acetate, pH 5.5 containing 5 mM EDTA and system 20 mM, in each case, optionally with 1 M DTT as stabilizer. The enzyme concentration used was 5 nM. The stock substrate solution was prepared as 10 mM in DMSO. Screenings were performed at a fixed substrate concentration of 60 pM and detailed kinetic studies with double substrate dilutions from 250 pM. The total concentration of DMSO in the trial remained below 3%. All tests were performed at room temperature. The fluorescence of the product (excitation at 390 nm, emission at 460 nm) was monitored with a fluorescent plate reader from Labsystems Fluoroskan Ascent. The product progress curves were generated more than 15 minutes after the generation of the AMC product.

Determination of the ki of cathepsin S

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The assay employs baculovirus-expressed human cathepsin S and the Boc-Val-Leu-Lys-AMC fluorescent substrate available in Bachem in a 384-well plate format, in which 7 test compounds can be tested in parallel with a positive control comprising a known comparison compound of cathepsin S inhibitor.

Dilutions of the substrate

280 pl / well of 12.5% DMSO was added to the B-H rows of two columns of a deep 96-well polypropylene plate. 70 pl / well of substrate was added to row A. 2 x 250 pl / well of assay buffer (100 mM Na phosphate, 10 mM NaCl, pH 6.5) was added to row A, mixed and mixed. double dilute along the plate to row H.

Inhibitor dilutions

100 pl / well of assay buffer is added to columns 2-5 and 7-12 of 4 rows of a 96-well V-bottom polypropylene plate. 200 pl / well of assay buffer are added to columns 1 and 6.

The first test compound prepared in DMSO is added to column 1 of the upper row, usually with a volume to provide between 10 and 30 times the approximate Ki, initially determined. The approximate Ki is calculated from a preliminary migration in which 10 pl / well of 1 mM boc-VLK-AMC (1/10 dilution of the 10 mM stock solution diluted in DMSo in assay buffer) is administered at rows from B to H and 20 pl / well to row A of a 96-well Microfluor® plate. 2 pl of each 10 mM test compound are added to a separate well in row A, columns 1-10. 90 pl of test buffer containing 1 mM DTT and S 2 nM cathepsin are added to each well of rows BH and 180 pl to row A. Row A is mixed with a multichannel pipette and diluted twice until row G. Row H is mixed and read in the fluorescent spectrophotometer. The readings are Prism data adjusted to the competitive inhibition equation, setting S = 100 pM and Km = 100 pM to obtain an estimate of Ki, up to a maximum of 100 pM.

The second test compound is added to column 6 of the upper row, the third to column 1 of the second row, etc. 1 pl of comparison compound is added to column 6 of the bottom row. Column 1 is mixed and diluted twice to column 5. Column 6 is mixed and diluted twice to column 10.

Using an 8-channel multistage pipette set at 5 x 10 pl, 10 pl / well of substrate is distributed to the 384-well test plate. The first column of the substrate dilution plate is distributed on all columns of the test plate, starting with row A. The space between the tips of the multichannel pipette will allow the alternate rows to be skipped correctly. The second column is distributed to all columns, starting in row B.

Using a 12-channel multi-stage pipette set at 4 x 10 pl, 10 pl / well of inhibitor is distributed to the 384-well test plate. The first row of the inhibitor dilution plate is distributed to the alternate rows of the test plate, starting at A1. The space between the tips of the multichannel pipette will allow the alternate columns to jump correctly. Similarly, the second, third and fourth rows are distributed to alternate rows and columns, starting at A2, B1 and B2, respectively.

20 ml of the assay buffer and 20 μl of 1 M DTT are mixed. Sufficient cathepsin S is added to provide a final concentration of 2 nM.

Using a distributor such as a Multidrop 384, 30 pl / well is added to all wells of the test plate and read in a fluorescence spectrophotometer such as an Ascent.

Fluorescence readings (excitation and emission wavelengths of 390 nm and 460 nm, respectively, established using bandpass filters) that reflect the degree of enzymatic cleavage of the fluorescent substrate, regardless of the inhibitor, are adjusted according to a linear rate for each well.

The adjusted rates for all wells for each inhibitor are adjusted to the competitive inhibition equation using SigmaPlot 2000 to determine the values of V, Km and Ki.

Cathepsin Ki L

The previous procedure, with the following modifications, is used to determine the Ki for cathepsin L.

The enzyme is commercially available human cathepsin L (eg, Calbiochem). The substrate is H-D-Val-Leu-Lys-AMC available in Bachem. The assay buffer is 100 mM sodium acetate 1 mM EDTA, pH 5.5). The stock solution in DMSO (10 mM in 100% DMSO) is diluted to 10% in assay buffer. The enzyme is prepared at a 5 nM concentration in assay buffer plus 1 mM dithiothreitol, just before use. 2 pl of 10 mM inhibitor prepared in 100% in DMSO are administered in row A. 10 pl of 50 pM substrate (= 1/200 dilution of 10 mM DMSO stock solution, diluted in assay buffer)

Inhibition studies

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Potential inhibitors are examined using the above assay with varying concentrations of the test compound. The reactions were initiated by the addition of enzyme to buffered substrate and inhibitor solutions. Ki values were calculated according to equation 1

image85

where Vo is the speed of the reaction, V is the maximum speed, S is the substrate concentration with a constant of Michaelis Km, and I is the inhibitor concentration.

The results are presented as

Less than 50 nanomolar

B 50-500 nanomolar

C 501-1000 nanomolar

D 1001 - 5000 nanomolar

E 5001 - 10,000 nanomolar

F more than 10,000 nanomolar

TABLE 1

 Example

 Catheter Ki K Cathepsin Ki S Cathepsin Ki L

 5

 A E C

 6

 A D C

 7

 A D D

 8

 A F D

 10

 A D B

 12

 A F D

 13

 A F D

The compounds of formula II are therefore potent inhibitors of cathepsin K and are still selective against cathepsins S and L, closely related.

Representative values of specific lots of compound / enzyme / test time include:

 Example

 Ki cathepsin K Ki cathepsin S Ki cathepsin L

 5

 1.6 7500 880

 6

 1.5 4000 890

 7

 4.2 1700 1200

 8

 3.3 13000 2900

 10

 3.4 4700 490

 12

 1.8 11000 1600

Metabolic stability

The compounds of the invention and the comparative examples indicated were tested for metabolic stability in a cytosolic assay in which the compounds were incubated with commercially available human hepatic cytosolic fractions and the disappearance of the compound was monitored by HPLC or LC / MS. . Clustered human liver cytosolic fractions are less likely to represent atypical individuals than

the blood of a single individual and can be stored frozen, unlike whole blood. Therefore, the cytosolic assay provides a test bench consisting of gma of the stability of a compound in the environment in vivo, such as when exposed to whole blood.

In summary, the test compounds (2 jM) are incubated in clustered human liver cytosol (Xenotech LLC 5 Lenexa US, 1 mg / ml protein in 0.1 M phosphate buffer, pH 7.4) at 37 ° C for A period of one hour. Incubations are initiated by the addition of 1 mM NADPH cofactor. Subsamples were taken at specific times at 0, 20, 40 and 60 minutes and "precipitated suddenly" by the addition of 3 volumes of acetonitrile cooled with ice. The samples were centrifuged at reduced temperature and the supernatants were separated and analyzed by LC-MS-MS.

10 Alternatively, an analogous stability test is performed on human or monkey whole blood.

Comparative example 3 uses the epimer with F down from unit P1 of WO0566180. Comparative example 2 employs the preferred units P1 and P2 of WO02 / 057270, together with a unit P3 within the scope of the present claims (which are outside the scope of WO02 / 057270). Comparative example 1 shows the epimer with Cl down the compound of Example 6.

15 TABLE 2

 Example

   ty2 minutes

 Comparative Example 2

 ___l H o N fi h n h it N \ o o 72

 Comparative Example 3

 1 F __l ^ H O \ ^ N-0 n 0 H ° S '\ 100-150

 Comparative Example 1

 1 Cl __ [? H o \ 0 f | H 11 H 11 N \ | \ L O O 24

 Example 6

 __1 Cl o \ 0 fl '^ 1 H If H Jl N \ O O> 300

 Example

   ty2 minutes

 Example 5

 1 Cl __l | H o \ S— \ 198

 Example 11

 and, ° o (rvH -r / \ H or H VyN ^ J 0 3 180

 Example 12

 F Cl. H i— \ / 'Y / ^ T ° \ —n \ JL) ° V 270

It will be apparent from comparative example 2 that P1 of the prior art of WO02 / 057270 provides compounds with a cytosolic half-life of a little more than one hour. P1 with the fluoro down broadly exemplified in WO05 / 66180 is somewhat better, with a half-life of more than 11 ^ hour. However, the replacement of chlorine with the downward fluorine of WO05 / 66180 dramatically reduces stability, as illustrated by comparing comparative example 1 with comparative example 3. In contrast, the The chlorine epimer of the invention (example 6) has provided a compound with a half-life of more than 5 hours. Similarly, with an identical component P3 and P2, comparative example 2 and the chlorine epimer above the invention (example 5) clearly demonstrate that the chlorine epimer upward 10 provides a much better half-life.

Permeability

This example measures the transport of inhibitors through human gastroenteric canal cells. The test uses the well-known Caco-2 cells with a number of passes between 40 and 60.

Apical transport to basolateral

In general, each compound will be tested in 2-4 wells. Basolateral and apical wells will contain 1.5 mL and 0.4 mL of transport buffer (TB), respectively, and the standard concentration of the test substances is 10 pM. In addition, all test solutions and buffers will contain 1% DMSO. Before the experiment, the transport plates are precoated with culture medium containing 10% serum for 30 minutes, to avoid non-specific binding to the plastic material. After 21 to 28 days of culture on 20 filter supports, the cells are ready for permeability experiments.

Transport plate No. 1 comprises 3 rows of 4 wells each. Row 1 is called washing, row 2 "30 minutes" and row 3 "60 minutes". The transport plate 2 comprises 3 rows of 4 wells, one is called row 4 "90 minutes", row 5 "120 minutes" and the remaining row is unassigned.

The culture medium of the apical wells is removed and the inserts are transferred to a washing row (# 1) on a transport plate (plate # 1) of the 2 plates without inserts, which have already been prepared with 1.5 mL of transport buffer (HBSS, 25 mM HEPES, pH 7.4) in rows 1 through 5. In the A ^ B screening the TB in the basolateral well also contains 1% bovine serum albumin.

0.5 mL of transport buffer (HBSS, 25 mM MONTH, pH 6.5) are added to the inserts and cell monolayers

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They are equilibrated in the transport buffer system for 30 minutes at 37 ° C on a Polymix shaker. After balancing with the buffer system, the transepithelial electrical resistance (TEER) value is measured in each well by an instrument with EVOm chopstick electrodes. TEER values generally comprise between 400 to 1000 Q per well (depending on the number of passes used).

The transport buffer (TB, pH 6.5) is removed from the apical part and the insert is transferred to the 30-minute row (# 2) and 425 pL of TB is added again (pH 6.5) , including the test substance to the apical well (donor). The plates are incubated on a Polymix shaker at 37 ° C with a low stirring speed of approximately 150 to 300 rpm.

After 30 minutes of incubation, in row 2 the inserts will be transferred to the new basolateral wells (receptors) previously heated, every 30 minutes; row 3 (60 minutes), 4 (90 minutes) and 5 (120 minutes).

Samples of 25 pL of the apical solution are taken after ~ 2 minutes and at the end of the experiment. These samples represent donor samples from the beginning and end of the experiment.

300 pL of the basolateral wells (receptors) will be taken at each scheduled time and the subsequent value of TEER at the end of the experiment is measured. Acetonitrile will be added to all collected samples until a final concentration of 50% in the samples. The collected samples will be stored at -20 ° C until analysis by HPLC or LC-MS.

Transportation from basolateral to apical

In general, each compound will be tested in 2-4 wells. The basolateral and apical wells will contain 1.55 mL and 0.4 mL of TB, respectively, and the standard concentration of the substances tested is 10 pM. In addition, all test solutions and buffers will contain 1% DMSO. Before the experiment, the transport plates are precoated with culture medium containing 10% serum for 30 minutes to avoid non-specific binding to the plastic material.

After 21 to 28 days of culture on filter supports, the cells are ready for permeability experiments. The culture medium of the apical wells is removed and the inserts are transferred to a washing row (# 1) in a new plate without inserts (transport plate).

The transport plate comprises 3 rows of 4 wells. Row 1 is called "washing" and row 3 is the "experimental row". The transport plate has been previously prepared with 1.5 mL of TB (pH 7.4) in wash row # 1 and with 1.55 mL of TB (pH 7.4), including the test substance, in experimental row n ° 3 (donor side).

0.5 mL of transport buffer (HBSS, 25 mM MES, pH 6.5) are added to the inserts in row # 1 and the cell monolayers are equilibrated in the transport buffer system for 30 minutes, 37 ° C on a Polymix shaker. After balancing with the buffer system, the TEER value is measured in each well using an EVOM chopstick electrode instrument.

The transport buffer (TB, pH 6.5) is removed from the apical part and the insert is transferred to row 3 and 400 pL of newly added TB, pH 6.5 is added to the inserts. After 30 minutes, 250 pL are removed from the apical well (receptor) and replaced with a new transport buffer. Thereafter, 250 pL of samples will be withdrawn and replaced with a new transport buffer, every 30 minutes until the end of the experiment at 120 minutes, and finally a subsequent TEER value is measured at the end of the experiment. A 25 pL sample will be taken from the basolateral (donor) compartment after ~ 2 minutes and at the end of the experiment. These samples represent donor samples from the beginning and end of the experiment.

Acetonitrile will be added to all collected samples until a final concentration of 50% in the samples. The collected samples will be stored at -20 ° C until analysis by HPLC or LC-MS.

Calculation

Determination of the accumulated fraction absorbed, FAcum, as a function of time. FAcum is calculated from:

FAcum =

where CRi is the concentration of receptor at the end of interval i and Coi is the concentration of donor at the beginning of interval i. A linear relationship must be obtained.

The determination of the permeability coefficients (Papp, cm / s) is calculated from:

image86

where k is the transport rate (min-1) defined as the slope obtained by linear regression of the absorbed cumulative fraction (FAcum) as a function of time (min), Vr is the volume in the receiving chamber (mL) and A is the area of the filter (cm2).

Typical reference compounds:

 Absorption category in man

 Markers% of absorption in man

 PASSIVE TRANSPORTATION

 Low (0-20%)

 Mannitol 16

 Methotrexate 20

 Moderate (21-75%)

 Acyclovir 30

 High (76-100%)

 Propranolol 90

 100 coffeema

 ACTIVE TRANSPORT

 Amino acid transporter

 L-Phenylalanine 100

 ACTIVE EFLUJO

 PGP-MDR1

 Digoxin 30

5

Representative results for the compounds of the invention in this Caco-2 assay include Papp values of 5.2 x 106 for the compound of Example 10 and 10 x 106 cm / s for the compound of Example 12. The compounds of formula IBs comprising a trifluoromethyl group, such as Example 13, generally have values of

 Papp 2-5 times higher.

 10

 Abbreviations

 DMF dimethylformamide DCM dichloromethane

 TBDMS ferc-butyldimethylsilyl TA room temperature

 THF tetrahydrofuran Ac acetyl

 TLC DMAP dimethylaminopyridine thin layer chromatography

 EtOAc ethyl acetate

Throughout this specification and in the claims that follow, unless the context indicates otherwise, the word "comprise" and variations such as "comprises" and "comprising" shall be understood to imply the inclusion of an integer, a stage, a group of whole numbers or a group of stages, but not the exclusion of any other whole number, stage, group of whole numbers or group of stages.

Claims (17)

5 10 fifteen image 1 1. A compound of Formula II: where R2 is the side chain of leucine, isoleucine, cyclohexylglycine, O-methyl threonine, 4-fluoroleucine or 3- methoxivaline; R3 is H, methyl or F; Rq is CF3 with the indicated stereochemistry and Rq 'is H; or Rq and Rq 'jointly define keto; What is it image2 where R4 is C1-C6 alkyl; R5 is H, methyl or F; R6 is C1-C6 alkyl; or a pharmaceutically acceptable salt, a hydrate or an N-oxide thereof. 2. The compound according to claim 1, with the formula Ila: twenty image3 where R2 is the side chain of leucine, isoleucine, cyclohexylglycine, O-methyl threonine, 4-fluoroleucine or 3- methoxivaline; R3 is H, methyl or F; 5 10 fifteen twenty 25 30 35 R4 is C1-C6 alkyl; R5 is H, methyl or F; or a pharmaceutically acceptable salt, a hydrate or an N-oxide thereof. 3. The compound according to claim 1 or 2, wherein R2 is the side chain of leucine, isoleucine, O-methyl threonine, 4-fluoroleucine or 3-methoxivaline. 4. The compound according to claim 3, wherein R2 is the 4-fluoroleucine or leucine side chain, preferably leucine. 5. The compound according to any one of claims 1 to 4, wherein R3 is fluoro or methyl, preferably in the meta position in relation to the holy amide. 6. The compound according to claim 5, wherein R3 is fluoro. 7. The compound according to any one of claims 1 to 4, wherein R3 is H. 8. The compound according to any one of claims 1 to 7, wherein R5 is fluoro. 9. The compound according to any one of claims 1 to 7, wherein R5 is H. 10. The compound according to any one of claims 1 to 9, wherein R4 is methyl. 11. The compound according to claim 1, selected from N- [1-6-Chloro-3-oxo-hexahydro-furo [3,2-5] pyrrol-4-carbonyl) -3-fluoro-3-methyl-butyl] -4- [2- (4-methyl -piperazin-1-il) - thiazol-4-yl] benzamide; N- [2- (6-Chloro-3-oxo-hexahydro-furo [3,2-6] pyrrol-4-yl) -1-cyclohexyl-2-oxo-ethyl] -4- [2- (4- methyl-piperazin-1-yl) -thiazol-4- il] -benzamide N- [1- (6-Chloro-3-oxo-hexahydro-furo [3,2-6] pyrrole-4-carbonyl) -3-methyl-butyl] -4- [2- (4-methyl-piperazin- 1-yl) -thiazol-4- il] benzamide N- [1- (6-Chloro-3-oxo-hexahydro-furo [3,2-b] pyrrole-4-carbonyl) -3-methyl-butyl] -4- [5-methyl-2- (4- methyl-piperazin-1-yl) -thiazol-4-yl] -benzamide; or a pharmaceutically acceptable salt, a hydrate or an N-oxide thereof. 12. The compound according to claim 1, termed N- [1- (6-Chloro-3-oxo-hexahydro-furo [3,2-b] pyrrole-4-carbonyl) -3-methyl-butyl] -3-fluoro-4- [2- (4- methyl-piperazin-1-yl) -thiazol-4-yl] -benzamide; or a pharmaceutically acceptable salt, a hydrate or an N-oxide thereof. 13. The compound according to claim 1, designated N- [1- (6-chloro-3-oxo-hexahydro-furo [3,2-b] pyrrole-4-carbonyl) -3-methyl-butyl] -4- [5-fluoro-2- (4-methyl-piperazin-1-yl) -thiazol-4-yl] -benzamide; or a pharmaceutically acceptable salt, a hydrate or an N-oxide thereof 14. The compound according to claim 1, with the formula lib image4 where R2 is the side chain of leucine, isoleucine, cyclohexylglycine, O-methyl threonine, 4-fluoroleucine or 3- methoxivaline; 5 10 fifteen twenty 25 R3 is H, methyl or F; Q is 4- (Ci-C4 alkyl) sulfonylphenyl; or a pharmaceutically acceptable salt, a hydrate or an N-oxide thereof. 15. The compound according to any one of claims 1 to 13, for use as a medicament. 16. Use of a compound as defined in any one of claims 1 to 14, in the preparation of a medicament for the treatment or prevention of a disorder selected from: osteoporosis, gingival diseases such as gingivitis and periodontitis, Paget's disease, hypercalcemia due to malignant disease, metabolic bone disease, diseases characterized by excessive degradation of the cartilage or matrix, such as osteoarthritis and rheumatoid arthritis, bone cancers that include neoplasia, pain. 17. The compound according to any one of claims 1 to 15, for use in the treatment or prevention of a disorder selected from: osteoporosis, gingival diseases such as gingivitis and periodontitis, Paget's disease, hypercalcemia due to malignant disease, metabolic bone disease, diseases characterized by excessive degradation of the cartilage or matrix, such as osteoarthritis and rheumatoid arthritis, bone cancers that include neoplasia, pain.